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| United States Patent |
6,093,728
|
|
McMahon
, et al.
|
July 25, 2000
|
Methods of modulating serine/threonine protein kinase function with
azabenzimidazole-based compounds
Abstract
The present invention is directed in part towards methods of modulating the
function of serine/threonine protein kinases with azabenzimidazole-based
compounds. The methods incorporate cells that express a serine/threonine
protein kinase, such as RAF. In addition, the invention describes methods
of preventing and treating serine/threonine protein kinase-related
abnormal conditions in organisms with a compound identified by the
invention. Furthermore, the invention pertains to azabenzimidazole
compounds and pharmaceutical compositions comprising these compounds.
| Inventors:
|
McMahon; Gerald (San Francisco, CA);
Weinberger; Heinz (Sulzbach/Ts, DE);
Kutscher; Bernhard (Maintal, DE);
App; Harald (San Francisco, CA)
|
| Assignee:
|
Asta Medica Aktiengesellschaft (Frankfurt, DE)
|
| Appl. No.:
|
160212 |
| Filed:
|
September 23, 1998 |
| Current U.S. Class: |
514/303; 546/118 |
| Intern'l Class: |
A61K 031/44; C07D 471/04 |
| Field of Search: |
546/118
514/303
|
References Cited
U.S. Patent Documents
| 3590045 | Jun., 1971 | Vogt et al. | 546/118.
|
| 4247556 | Jan., 1981 | Von Bebenburg | 544/362.
|
| 5217999 | Jun., 1993 | Levitzki et al. | 514/613.
|
| 5302606 | Apr., 1994 | Spada et al. | 514/357.
|
| 5330992 | Jul., 1994 | Eissenstat et al. | 514/312.
|
| Foreign Patent Documents |
| 0 566 226 | Oct., 1993 | EP.
| |
| 91/15495 | Oct., 1991 | WO.
| |
| 92/20642 | Nov., 1992 | WO.
| |
| 92/21660 | Dec., 1992 | WO.
| |
| 94/03427 | Feb., 1994 | WO.
| |
| 94/14808 | Jul., 1994 | WO.
| |
| 96/22976 | Aug., 1996 | WO.
| |
| 96/40116 | Dec., 1996 | WO.
| |
Other References
Bonner et al., "Structure and Biological Activity of Human Homologs of the
raf/mil Oncogene," Molecular and Cellular Biology 5:1400-1407 (1985).
Folkman, "What is the Evidence that Tumors are Angiogenesis Dependent?"
Journal of the National Cancer Institute 82:4-6 (1990).
Monia et al., "Antitumor activity of a phosphorothioate antisense
oligodeoxynucleotide targeted against C-raf kinase," Nature Medicine
2:668-675 (1996).
Morrison et al., "Signal Transduction From Membrane to Cytoplasm: Growth
Factors and Membrane-Bound Oncogene Products Increase Raf-1
Phosphorylation and Associated Protein Kinase Activity," Proc. Natl. Acad.
Sci. USA 85:8855-8859 (1988).
Robbins et al., "Regulation and properties of extracellular
signal-regulated protein kinases 1 and 2 in vitro," J. Biol. Chem.
268:5097-5106 (1993).
|
Primary Examiner: Dentz; Bernard
Attorney, Agent or Firm: Pillsbury Madison & Sutro, LLP
Parent Case Text
RELATED APPLICATIONS
This application claims priority to the U.S. Provisional Application No.
60/060,145, filed Sep. 26, 1997, by McMahan et al., and entitled "METHODS
OF MODULATING SERINE/THREONINE PROTEIN KINASE FUNCTION WITH
AZABENZIMIDAZOLE-BASED COMPOUNDS", which is hereby incorporated by
reference in its entirety herein, including any drawings.
Claims
What is claimed is:
1. An azabenzimidazole compound having a structure set forth in formula II,
or III:
##STR6##
wherein (a) R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are independently
selected from the group consisting of
(i) hydrogen;
(ii) saturated or unsaturated alkyl;
(iii) NX.sub.2 X.sub.3, where X.sub.2 and X.sub.3 are independently
selected from the group consisting of hydrogen, saturated or unsaturated
alkyl, and homocyclic or heterocyclic ring moieties;
(iv) halogen and trihalomethyl;
(v) a ketone of formula --CO--X.sub.4, where X.sub.4 is selected from the
group consisting of hydrogen, alkyl, and homocyclic or heterocyclic ring
moieties;
(vi) a carboxylic acid of formula --(X.sub.5).sub.n --COOH or ester of
formula --(X.sub.6).sub.n --COO--X.sub.7, where X.sub.5, X.sub.6, and
X.sub.7 and are independently selected from the group consisting of alkyl
and homocyclic or heterocyclic ring moieties and where n is 0 or 1;
(vii) an alcohol of formula (X.sub.8).sub.n --OH or an alkoxy moiety of
formula --(X.sub.8).sub.n --O--X.sub.9, where X.sub.8 and X.sub.9 are
independently selected from the group consisting of hydrogen, saturated or
unsaturated alkyl, and homocyclic or heterocyclic ring moieties, wherein
said ring is optionally substituted with one or more substituents
independently selected from the group consisting of alkyl, alkoxy,
halogen, trihalomethyl, carboxylate, nitro, and ester and where n is 0 or
1;
(viii) an amide of formula --NHCOX.sub.10, where X.sub.10 is selected from
the group consisting of alkyl, hydroxyl, and homocyclic or heterocyclic
ring moieties, wherein said ring is optionally substituted with one or
more substituents independently selected from the group consisting of
alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester;
(ix) --SO.sub.2 NX.sub.11 X.sub.12, where X.sub.11 and X.sub.12 are
selected from the group consisting of hydrogen, alkyl, and homocyclic or
heterocyclic ring moieties;
(x) a homocyclic or heterocyclic ring moiety optionally substituted with
one, two, or three substituents independently selected from the group
consisitng of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro,
and ester moieties;
(xi) an aldehyde of formula --CO--H; and
(xii) a sulfone of formula --SO.sub.2 --X.sub.13, where X.sub.13 is
selected from the group consisting of saturated or unsaturated alkyl and
homocyclic or heterocyclic ring moieties;
(b) Z.sub.1 and Z.sub.2 are independently selected from the group
consisting of nitrogen, NH and NR.sub.4 ; and
(c) Z.sub.3 and X.sub.1 are independently selected from the group
consisting of NH, sulfur, and oxygen.
2. The compound of claim 1, wherein said Z.sub.1 and Z.sub.2 are
independently selected from the group consisting of nitrogen and NH.
3. The compound of claim 2, wherein said R.sub.1, R.sub.2, R.sub.3, and
R.sub.4 are independently selected from the group consisting of
(i) hydrogen;
(ii) saturated or unsaturated alkyl optionally substituted with a
homocyclic or heterocyclic ring moiety, or a polycyclic ring moiety,
wherein said ring moiety is optionally substituted with one, two, or three
substituents independently selected from the group consisting of alkyl,
halogen, trihalomethyl, hydroxy, alkoxy, carboxylate, nitro, and ester
moieties; and
(iii) a homocyclic or heterocyclic ring moiety optionally substituted with
one, two, or three substituents independently selected from the group
consisting of alkyl, halogen, trihalomethyl, hydroxy, alkoxy, carboxylate,
nitro, and ester moieties.
4. The compound of claim 3, wherein said R.sub.2 and R.sub.3 are hydrogen.
5. The compound of claim 4, wherein said R.sub.1 is phenyl optionally
substituted with one, two, or three substituents independently selected
from the group consisting of alkyl, alkoxy, halogen, trihalomethyl,
carboxylate, nitro, or ester moieties.
6. The compound of claim 5, wherein said R.sub.1 is selected from the group
consisting of SABI substituents.
7. The compound of claim 6, wherein said X.sub.1 is selected from the group
consisting of sulfur, oxygen, and NH.
8. The compound of claim 7, wherein said Z.sub.3 is oxygen.
9. The compound of claim 8, wherein said R.sub.4 is selected from the group
consisting of methyl and ethyl.
10. The compound of claim 9, wherein said azabenzimidazole compound is
selected from the group consisting of SABI compounds.
11. A pharmaceutical composition comprising an azabenzimidazole compound of
any one of claims 1-10, and a physiologically acceptable carrier or
diluent.
Description
BACKGROUND OF THE INVENTION
The following description of the background of the invention is provided to
aid in understanding the invention but is not admitted to be prior art to
the invention.
Cellular signal transduction is a fundamental mechanism whereby external
stimuli regulating diverse cellular processes are relayed to the interior
of cells. One of the key biochemical mechanisms of signal transduction
involves the reversible phosphorylation of proteins, which enables
regulation of the activity of mature proteins by altering their structure
and function.
The best characterized protein kinases in eukaryotes phosphorylate proteins
on the alcohol moiety of serine, threonine, and tyrosine residues. These
kinases largely fall into two groups, those specific for phosphorylating
serine and threonine, and those specific for phosphorylating tyrosine.
Some kinases, referred to as "dual specificity" kinases, are able to
phosphorylate on tyrosine as well as serine/threonine residues.
Protein kinases can also be characterized by their location within the
cell. Some kinases are transmembrane receptor proteins capable of binding
ligands external to the cell membrane. Binding the ligands alters the
receptor protein kinase's catalytic activity. Others are non-receptor
proteins lacking a transmembrane domain. Non-receptor protein kinases can
be found in a variety of cellular compartments from the inner-surface of
the cell membrane to the nucleus.
Many kinases are involved in regulatory cascades where their substrates may
include other kinases whose activities are regulated by their
phosphorylation state. Ultimately the activity of a downstream effector is
modulated by phosphorylation resulting from activation of such a pathway.
The serine/threonine kinase family includes members that regulate many
steps of signaling cascades, including cascades controlling cell growth,
migration, differentiation, gene expression, muscle contraction, glucose
metabolism, cellular protein synthesis, and regulation of the cell cycle.
An example of a non-receptor protein kinase that phosphorylates protein
targets on serine and threonine residues is RAF. RAF modulates the
catalytic activity of other protein kinases, such as the protein kinase
that phosphorylates and thereby activates mitogen activated protein kinase
(MAPK). RAF itself is activated by the membrane anchored protein RAS,
which in turn is activated in response to ligand activated tyrosine
receptor protein kinases such as epidermal growth factor receptor (EGFR)
and platelet-derived growth factor receptor (PDGFR). The biological
importance of RAF in controlling cellular events is underscored by the
finding that altered forms of RAF can cause cancer in organisms. Evidence
for importance of RAF in malignancies is provided in Monia et al., 1996,
Nature Medicine 2: 668, incorporated herein by reference in its entirety
including all figures and tables.
In an effort to discover novel treatments for cancer and other diseases,
biomedical researchers and chemists have designed, synthesized, and tested
molecules that inhibit the function of protein kinases. Some small organic
molecules form a class of compounds that modulate the function of protein
kinases. Examples of molecules that have been reported to inhibit the
function of protein kinases are bis monocyclic, bicyclic or heterocyclic
aryl compounds (PCT WO 92/20642), vinylene-azaindole derivatives (PCT WO
94/14808), 1-cyclopropyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992),
styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridyl
compounds (U.S. Pat. No. 5,302,606), certain quinazoline derivatives (EP
Application No. 0 566 266 A1), seleoindoles and selenides (PCT WO
94/03427), tricyclic polyhydroxylic compounds (PCT WO 92/21660), and
benzylphosphonic acid compounds (PCT WO 91/15495).
The compounds that can traverse cell membranes and are resistant to acid
hydrolysis are potentially advantageous therapeutics as they can become
highly bioavailable after being administered orally to patients. However,
many of these protein kinase inhibitors only weakly inhibit the function
of protein kinases. In addition, many inhibit a variety of protein kinases
and will therefore cause multiple side-effects as therapeutics for
diseases.
SUMMARY OF THE INVENTION
The present invention is directed in part towards methods of modulating the
function of serine/threonine protein kinases with azabenzimidazole-based
compounds. The methods incorporate cells that express a serine/threonine
protein kinase, such as RAF. In addition, the invention describes methods
of preventing and treating serine/threonine protein kinase-related
abnormal conditions in organisms with a compound identified by the
invention. Furthermore, the invention pertains to pharmaceutical
compositions comprising compounds identified by methods of the invention.
I. Methods for Screening Compounds that Modulate Serine/Threonine Protein
Kinase Function
The methods of the present invention provide means for modulating the
function of both receptor and cytosolic serine/threonine protein kinases.
These methods provide means of modulating the enzymes both in vitro and in
vivo. For in vitro applications, the methods of the invention relate in
part to method of identifying compounds that modulate the function of
serine/threonine protein kinases.
Thus, in a first aspect, the invention features a method of modulating the
function of a serine/threonine protein kinase with an
azabenzimidazole-based compound. The azabenzimidazole compound is
optionally substituted with organic groups. The method comprises comprises
contacting cells expressing the serine/threonine protein kinase with the
compound.
The term "function" refers to the cellular role of a serine/threonine
protein kinase. The serine/threonine protein kinase family includes
members that regulate many steps in signaling cascades, including cascades
controlling cell growth, migration, differentiation, gene expression,
muscle contraction, glucose metabolism, cellular protein synthesis, and
regulation of the cell cycle.
The term "modulates" refers to the ability of a compound to alter the
function of a protein kinase. A modulator preferably activates the
catalytic activity of a protein kinase, more preferably activates or
inhibits the catalytic activity of a protein kinase depending on the
concentration of the compound exposed to the protein kinase, or most
preferably inhibits the catalytic activity of a protein kinase.
The term "catalytic activity", in the context of the invention, defines the
rate at which a protein kinase phosphorylates a substrate. Catalytic
activity can be measured, for example, by determining the amount of a
substrate converted to a product as a function of time. Phosphorylation of
a substrate occurs at the active-site of a protein kinase. The active-site
is normally a cavity in which the substrate binds to the protein kinase
and is phosphorylated.
The term "substrate" as used herein refers to a molecule phosphorylated by
a serine/threonine protein kinase. The substrate is preferably a peptide
and more preferably a protein. In relation to the protein kinase RAF,
preferred substrates are MEK and the MEK substrate MAPK.
The term "activates" refers to increasing the cellular function of a
protein kinase. The protein kinase function is preferably the interaction
with a natural binding partner and most preferably catalytic activity.
The term "inhibit" refers to decreasing the cellular function of a protein
kinase. The protein kinase function is preferably the interaction with a
natural binding partner and most preferably catalytic activity.
The term "modulates" also refers to altering the function of a protein
kinase by increasing or decreasing the probability that a complex forms
between a protein kinase and a natural binding partner. A modulator
preferably increases the probability that such a complex forms between the
protein kinase and the natural binding partner, more preferably increases
or decreases the probability that a complex forms between the protein
kinase and the natural binding partner depending on the concentration of
the compound exposed to the protein kinase, and most preferably decreases
the probability that a complex forms between the protein kinase and the
natural binding partner.
The term "complex" refers to an assembly of at least two molecules bound to
one another. Signal transduction complexes often contain at least two
protein molecules bound to one another. For instance, a protein tyrosine
receptor protein kinase, GRB2, SOS, RAF, and RAS assemble to form a signal
transduction complex in response to a mitogenic ligand.
The term "natural binding partner" refers to polypeptides that bind to a
protein kinase in cells. Natural binding partners can play a role in
propagating a signal in a protein kinase signal transduction process. A
change in the interaction between a protein kinase and a natural binding
partner can manifest itself as an increased or decreased probability that
the interaction forms, or an increased or decreased concentration of the
protein kinase/natural binding partner complex.
A protein kinase natural binding partner can bind to a protein kinase's
intracellular region with high affinity. High affinity represents an
equilibrium binding constant on the order of 10.sup.-6 M or less. In
addition, a natural binding partner can also transiently interact with a
protein kinase intracellular region and chemically modify it. Protein
kinase natural binding partners are chosen from a group that includes, but
is not limited to, SRC homology 2 (SH2) or 3 (SH3) domains, other
phosphoryl tyrosine binding (PTB) domains, guanine nucleotide exchange
factors, protein phosphatases, and other protein kinases. Methods of
determining changes in interactions between protein kinases and their
natural binding partners are readily available in the art.
The term "serine/threonine protein kinase" refers to an enzyme with an
amino acid sequence with at least 10% amino acid identity to other enzymes
that phosphorylate proteins on serine and threonine residues. A
serine/threonine protein kinase catalyzes the addition of phosphate onto
proteins on serine and threonine residues. Serine/threonine protein
kinases can exist as membrane bound proteins or cytosolic proteins.
The term "contacting" as used herein refers to mixing a solution comprising
an azabenzimidazole compound of the invention with a liquid medium bathing
the cells of the methods. The solution comprising the compound may also
comprise another component, such as dimethylsulfoxide (DMSO), which
facilitates the uptake of the azabenzimidazole compound or compounds into
the cells of the methods. The solution comprising the azabenzimidazole
compound may be added to the medium bathing the cells by utilizing a
delivery apparatus, such as a pipet-based device or syringe-based device.
The term "azabenzimidazole-based compound" refers to an azabenzimidazole
organic compound substituted with chemical substituents. Azabenzimidazole
compounds are of the general structure:
##STR1##
The term "substituted", in reference to the invention, refers to an
azabenzimidazole compound that is derivatized with any number of chemical
substituents.
In a preferred embodiment, the invention relates to the method of
modulating the function of a serine/threonine protein kinase, where the
protein kinase is RAF.
The RAF protein kinase phosphorylates protein targets on serine or
threonine residues. One such protein target is the protein kinase (MEK)
that phosphorylates and consequently activates mitogen activated protein
kinase (MAPK). RAF itself is activated by the membrane-bound guanine
triphosphate hydrolyzing enzyme RAS in response to mitogen-stimulated
receptor protein tyrosine kinases, such as epidermal growth factor
receptor (EGFR) and platelet-derived growth factor receptor (PDGFR).
The methods of the present invention can detect compounds that modulate the
function of the RAF protein kinase in cells. RAF phosphorylates a protein
kinase (MEK) which in turn phosphorylates mitogen-activated protein kinase
(MAPK). Assays that monitor only the phosphorylation of MEK by RAF are not
sensitive because the phosphorylation levels of MEK are not significant.
To overcome this sensitivity dilemma, the phosphorylation of both MEK and
MAPK are followed in the assays of the present invention. The MAPK
phosphorylation signal amplifies the MEK phosphorylation signal and allows
RAF-dependent phosphorylation to be followed in assays, such as
enzyme-linked immunosorbant assays. In addition, the assay of the
invention is performed in a high throughput format such that many
compounds can be rapidly monitored in a short period of time.
In another aspect, the invention descries a method of identifying compounds
that modulate the function of serine/threonine protein kinase, comprising
the steps of contacting cells expressing the serine/threonine protein
kinase with the compound, and monitoring an effect upon the cells.
The term "monitoring" refers to observing the effect of adding the compound
to the cells of the method. The effect can be manifested in a change in
cell phenotype, cell proliferation, protein kinase catalytic activity, or
in the interaction between a protein kinase and a natural binding partner.
The term "effect" describes a change or an absence of a change in cell
phenotype or cell proliferation. "Effect" can also describe a change or an
absence of a change in the catalytic activity of the protein kinase.
"Effect" can also describe a change or an absence of a change in an
interaction between the protein kinase and a natural binding partner.
A preferred embodiment of the invention relates to the method of
identifying compounds that modulate the function of serine/threonine
protein kinase, where the effect is a change or an absence of a change in
cell phenotype.
The term "cell phenotype" refers to the outward appearance of a cell or
tissue or the function of the cell or tissue. Examples of cell phenotype
are cell size (reduction or enlargement), cell proliferation (increased or
decreased numbers of cells), cell differentiation (a change or absence of
a change in cell shape), cell survival, apoptosis (cell death), or the
utilization of a metabolic nutrient (e.g., glucose uptake). Changes or the
absence of changes in cell phenotype are readily measured by techniques
known in the art.
In another preferred embodiment, the invention relates to the method of
identifying compounds that modulate the function of serine/threonine
protein kinase, where the effect is a change or an absence of a change in
cell proliferation.
The term "cell proliferation" refers to the rate at which a group of cells
divides. The number of cells growing in a vessel can be quantified by a
person skilled in the art when that person visually counts the number of
cells in a defined volume using a common light microscope. Alternatively,
cell proliferation rates can be quantified by laboratory apparatae that
optically or conductively measure the density of cells in an appropriate
medium.
In another preferred embodiment, the invention relates to the method of
identifying compounds that modulate the function of serine/threonine
protein kinase, where the effect is a change or an absence of a change in
the interaction between the serine/threonine protein kinase with a natural
binding partner.
The term "interaction", in the context of the invention, describes a
complex formed between a protein kinase's intracellular region and a
natural binding partner or compound. The term "interaction" can also
extend to a complex formed between a compound of the invention with
intracellular regions and extracellular regions of the protein kinase
under study. Although a cytosolic protein kinase will have no
extracellular region, a receptor protein kinase will harbor both an
extracellular and an intracellular region.
The term "intracellular region" as used herein refers to the portion of a
protein kinase which exists inside a cell. The term "extracellular region"
as used herein refers to a portion of a protein kinase which exists
outside of the cell.
In a preferred embodiment, the invention relates to the method of
identifying compounds that modulate the function of serine/threonine
protein kinase that further comprises the following steps: (a) lysing the
cells to render a lysate comprising serine/threonine protein kinase; (b)
absorbing the serine/threonine protein kinase to an antibody; (c)
incubating the adsorbed serine/threonine protein kinase with a substrate
or substrates; and (d) adsorbing the substrate or substrates to a solid
support or antibody. The step of monitoring the effect on the cells
comprises measuring the phosphate concentration of the substrate or
substrates.
The term "lysing" as used herein refers to a method of disrupting the
integrity of a cell such that its interior contents are liberated. Cell
lysis is accomplished by many techniques known to persons skilled in the
art. The method is accomplished preferably by sonication or cell sheering
techniques and more preferably by detergent techniques.
The term "antibody" as used herein refers to a protein molecule that
specifically binds a protein kinase. An antibody preferably binds to one
class of protein kinase and more preferably specifically binds to the RAF
protein kinase.
The term "specifically binds" as used herein refers to an antibody that
binds a protein kinase with higher affinity than another protein kinase or
cellular protein. An antibody that specifically binds to a protein kinase
will bind a higher concentration of the specific protein kinase than any
other protein kinase or cellular protein.
The term "adsorbing" as used herein refers to the binding of a molecule to
the surface of an antibody or solid support. Examples of solid supports
are chemically modified cellulose, such as phosphocellulose, and nylon.
Antibodies can be linked to solid supports using techniques well known to
individuals of ordinary skill in the art. See, e.g., Harlo & Lane,
Antibodies, A Laboratory Manual, 1989, Cold Spring Harbor Laboratories.
The term "measuring the phosphate concentration" as used herein refers to
techniques commonly known to persons of ordinary skill in the art. These
techniques can involve quantifying the concentration of phosphate
concentrations within a substrate or determining relative amounts of
phosphate within a substrate. These techniques can include adsorbing the
substrate to a membrane and detecting the amount of phosphate within the
substrate by radioactive measurements.
In another preferred embodiment, the invention relates to the method of
identifying compounds that modulate the function of serine/threonine
protein kinase that further comprises the following steps: (a) lysing the
cells to render a lysate comprising RAF; (b) adsorbing the RAF to an
antibody; (c) incubating the adsorbed RAF with MEK and MAPK; and (d)
adsorbing the MEK and MAPK to a solid support or antibody or antibodies.
The step of measuring the effect on the cells comprises monitoring the
phosphate concentration of said MEK and MAPK.
In a preferred embodiment, the invention relates to the method of
identifying compounds that modulate the function of serine/threonine
protein kinase, where the azabenzimidazole-based compound has a structure
set forth in formula I, II, or III as defined herein or any of the
subgroups thereof set forth herein.
The term "compound" refers to the compound or a pharmaceutically acceptable
salt, ester, amide, prodrug, isomer, or metabolite, thereof.
The term "pharmaceutically acceptable salt" refers to a formulation of a
compound that does not abrogate the biological activity and properties of
the compound. Pharmaceutical salts can be obtained by reacting a compound
of the invention with inorganic or organic acids such as hydrochloric
acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid,
methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,
salicylic acid and the like.
The term "prodrug" refers to an agent that is converted into the parent
drug in vivo. Prodrugs may be easier to administer than the parent drug in
some situations. For example, the prodrug may be bioavailable by oral
administration but the parent is not, or the prodrug may improve
solubility to allow for intravenous administration.
In another preferred embodiment, the invention relates to the method of
identifying compounds that modulate the function of serine/threonine
protein kinase, where the azabenzimidazole-based compound has a structure
set forth in formula I, II, or III, where the azabenzimidazole compound is
selected from the group consisting of SABI compounds.
The term "SABI compounds" refers to the group of azabenzimidazole-based
compounds having a structure set forth in formula A or B, and numbered A-1
through A-198 in the following table:
______________________________________
(A)
#STR2##
(B)
##STR3##
-
Compound
No. R.sub.1 X.sub.1 R.sub.4 Z.sub.3
______________________________________
A-1 phenyl O -- --
A-2 phenyl S -- --
A-3 phenyl O methyl O
A-4 phenyl O ethyl O
A-5 2-nitrophenyl O -- --
A-6 3-nitrophenyl O -- --
A-7 4-nitrophenyl O -- --
A-8 2-chlorophenyl O -- --
A-9 3-chlorophenyl O -- --
A-10 4-chlorophenyl O -- --
A-11 2-methylphenyl O -- --
A-12 3-methylphenyl O -- --
A-13 4-methylphenyl O -- --
A-14 2-fluorophenyl O -- --
A-15 3-fluorophenyl O -- --
A-16 4-fluorophenyl O -- --
A-17 2-(trifluoromethyl)phenyl O -- --
A-18 3-(trifluoromethyl)phenyl O -- --
A-19 4-(trifluoromethyl)phenyl O -- --
A-20 2-methoxyphenyl O -- --
A-21 3-methoxyphenyl O -- --
A-22 4-methoxyphenyl O -- --
A-23 2-carboxyphenyl O -- --
A-24 3-carboxyphenyl O -- --
A-25 4-carboxyphenyl O -- --
A-26 2-nitrophenyl S -- --
A-27 3-nitrophenyl S -- --
A-28 4-nitrophenyl S -- --
A-29 2-chlorophenyl S -- --
A-30 3-chlorophenyl S -- --
A-31 4-chlorophenyl S -- --
A-32 2-methylphenyl S -- --
A-33 3-methylphenyl S -- --
A-34 4-methylphenyl S -- --
A-35 2-fluorophenyl S -- --
A-36 3-fluorophenyl S -- --
A-37 4-fluorophehyl S -- --
A-38 2-(trifluoromethyl)phenyl S -- --
A-39 3-(trifluoromethyl)phenyl S -- --
A-40 4-(trifluoromethyl)phenyl S -- --
A-41 2-methoxyphenyl S -- --
A-42 3-methoxyphenyl S -- --
A-43 4-methoxyphenyl S -- --
A-44 2-carboxyphenyl S -- --
A-45 3-carboxyphenyl S -- --
A-46 4-carboxyphenyl S -- --
A-47 phenyl NH -- --
A-48 2-nitrophenyl NH -- --
A-49 3-nitrophenyl NH -- --
A-50 4-nitrophenyl NH -- --
A-51 2-chlorophenyl NH -- --
A-52 3-chlorophenyl NH -- --
A-53 4-chlorophenyl NH -- --
A-54 2-methylphenyl NH -- --
A-55 3-methylphenyl NH -- --
A-56 4-methylphenyl NH -- --
A-57 2-fluorophenyl NH -- --
A-58 3-fluorophenyl NH -- --
A-59 4-fluorophenyl NH -- --
A-60 2-(trifluoromethyl)phenyl NH -- --
A-61 3-(trifluoromethyl)phenyl NH -- --
A-62 4-(trifluoromethyl)phenyl NH -- --
A-63 2-methoxyphenyl NH -- --
A-64 3-methoxyphenyl NH -- --
A-65 4-methoxyphenyl NH -- --
A-66 2-carboxyphenyl NH -- --
A-67 3-carboxyphenyl NH -- --
A-68 4-carboxyphenyl NH -- --
A-69 2-nitrophenyl O methyl O
A-70 3-nitrophenyl O methyl O
A-71 4-nitrophenyl O methyl O
A-72 2-chlorophenyl O methyl O
A-73 3-chlorophenyl O methyl O
A-74 4-chlorophenyl O methyl O
A-75 2-methylphenyl O methyl O
A-76 3-methylphenyl O methyl O
A-77 4-methylphenyl O methyl O
A-78 2-fluorophenyl O methyl O
A-79 3-fluorophenyl O methyl O
A-80 4-fluorophenyl O methyl O
A-81 2-(trifluoromethyl)phenyl O methyl O
A-82 3-(trifluoromethyl)phenyl O methyl O
A-83 4-(trifluoromethyl)phenyl O methyl O
A-84 2-methoxyphenyl O methyl O
A-85 3-methoxyphenyl O methyl O
A-86 4-methoxyphenyl O methyl O
A-87 2-carboxyphenyl O methyl O
A-88 3-carboxyphenyl O methyl O
A-89 4-carboxyphenyl O methyl O
A-90 phenyl S methyl O
A-91 2-nitrophenyl S methyl O
A-92 3-nitrophenyl S methyl O
A-93 4-nitrophenyl S methyl O
A-94 2-chlorophenyl S methyl O
A-95 3-chlorophenyl S methyl O
A-96 4-chlorophenyl S methyl O
A-97 2-methylphenyl S methyl O
A-98 3-methylphenyl S methyl O
A-99 4-methylphenyl S methyl O
A-100 2-fluorophenyl S methyl O
A-101 3-fluorophenyl S methyl O
A-102 4-fluorophenyl S methyl O
A-103 2-(trifluoromethyl)phenyl S methyl O
A-104 3-(trifluoromethyl)phenyl S methyl O
A-105 4-(trifluoromethyl)phenyl S methyl O
A-106 2-methoxyphenyl S methyl O
A-107 3-methoxyphenyl S methyl O
A-108 4-methoxyphenyl S methyl O
A-109 2-carboxyphenyl S methyl O
A-110 3-carboxyphenyl S methyl O
A-111 4-carboxyphenyl S methyl O
A-112 phenyl NH methyl O
A-113 2-nitrophenyl NH methyl O
A-114 3-nitrophenyl NH methyl O
A-115 4-nitrophenyl NH methyl O
A-116 2-chlorophenyl NH methyl O
A-117 3-chlorophenyl NH methyl O
A-118 4-chlorophenyl NH methyl O
A-119 2-methylphenyl NH methyl O
A-120 3-methylphenyl NH methyl O
A-121 4-methylphenyl NH methyl O
A-122 2-fluorophenyl NH methyl O
A-123 3-fluorophenyl NH methyl O
A-124 4-fluorophenyl NH methyl O
A-125 2-(trifluoromethyl)phenyl NH methyl O
A-126 3-(trifluoromethyl)phenyl NH methyl O
A-127 4-(trifluorometbyl)phenyl NH methyl O
A-128 2-methoxyphenyl NH methyl O
A-129 3-methoxyphenyl NH methyl O
A-130 4-methoxyphenyl NH methyl O
A-131 2-carboxyphenyl NH methyl O
A-132 3-carboxyphenyl NH methyl O
A-133 4-carboxyphenyl NH methyl O
A-134 2-nitrophenyl O ethyl O
A-135 3-nitrophenyl O ethyl O
A-136 4-nitrophenyl O ethyl O
A-137 2-chlorophenyl O ethyl O
A-138 3-chlorophenyl O ethyl O
A-139 4-chlorophenyl O ethyl O
A-140 2-methylphenyl O ethyl O
A-141 3-methylphenyl O ethyl O
A-142 4-methylphenyl O ethyl O
A-143 2-fluorophenyl O ethyl O
A-144 3-fluorophenyl O ethyl O
A-145 4-fluorophenyl O ethyl O
A-146 2-(trifluoromethyl)phenyl O ethyl O
A-147 3-(trifluoromethyl)phenyl O ethyl O
A-148 4-(trifluoromethyl)phenyl O ethyl O
A-149 2-methoxyphenyl O ethyl O
A-150 3-methoxyphenyl O ethyl O
A-151 4-methoxyphenyl O ethyl O
A-152 2-carboxyphenyl O ethyl O
A-153 3-carboxyphenyl O ethyl O
A-154 4-carboxyphenyl O ethyl O
A-155 phenyl S ethyl O
A-156 2-nitrophenyl S ethyl O
A-157 3-nitrophenyl S ethyl O
A-158 4-nitrophenyl S ethyl O
A-159 2-chlorophenyl S ethyl O
A-160 3-chlorophenyl S ethyl O
A-161 4-chlorophenyl S ethyl O
A-162 2-methylphenyl S ethyl O
A-163 3-methylphenyl S ethyl O
A-164 4-methylphenyl S ethyl O
A-165 2-fluorophenyl S ethyl O
A-166 3-fluorophenyl S ethyl O
A-167 4-fluorophenyl S ethyl O
A-168 2-(trifluoromethyl)phenyl S ethyl O
A-169 3-(trifluoromethyl)phenyl S ethyl O
A-170 4-(trifluoromethyl)phenyl S ethyl O
A-171 2-methoxyphenyl S ethyl O
A-172 3-methoxyphenyl S ethyl O
A-173 4-methoxyphenyl S ethyl O
A-174 2-carboxyphenyl S ethyl O
A-175 3-carboxyphenyl S ethyl O
A-176 4-carboxyphenyl S ethyl O
A-177 phenyl NH ethyl O
A-178 2-nitrophenyl NH ethyl O
A-179 3-nitrophenyl NH ethyl O
A-180 4-nitrophenyl NH ethyl O
A-181 2-chlorophenyl NH ethyl O
A-182 3-chlorophenyl NH ethyl O
A-183 4-chlorophenyl NH ethyl O
A-184 2-methylphenyl NH ethyl O
A-185 3-methylphenyl NH ethyl O
A-186 4-methylphenyl NH ethyl O
A-187 2-fluorophenyl NH ethyl O
A-188 3-fluorophenyl NH ethyl O
A-189 4-fluorophenyl NH ethyl O
A-190 2-(trifluoromethyl)phenyl NH ethyl O
A-191 3-(trifluoromethyl)phenyl NH ethyl O
A-192 4-(trifluoromethyl)phenyl NH ethyl O
A-193 2-methoxyphenyl NH ethyl O
A-194 3-methoxyphenyl NH ethyl O
A-195 4-methoxyphenyl NH ethyl O
A-196 2-carboxyphenyl NH ethyl O
A-197 3-carboxyphenyl NH ethyl O
A-198 4-carboxyphenyl NH ethyl O
______________________________________
II. Methods of Preventing or Treating Abnormal Conditions
In another aspect, the invention features a method of preventing or
treating an abnormal condition in an organism by administering a compound
of the invention, as specified herein by formula I, II, or III, with any
of the constraints provided herein, to an organism.
The term "organism" relates to any living entity comprising at least one
cell. An organism can be as simple as one eukaryotic cell or as complex as
a mammal. In preferred embodiments, an organism refers to humans or
mammals.
The term "preventing" refers to the method of the invention decreasing the
probability, or eliminating the possibility, that an organism contracts or
develops the abnormal condition.
The term "treating" refers to the method of the invention having a
therapeutic effect and at least partially alleviating or abrogating the
abnormal condition in the organism.
The term "therapeutic effect" refers to the inhibition of cell growth
causing or contributing to an abnormal condition. The term "therapeutic
effect" also refers to the inhibition of growth factors causing or
contributing to the abnormal condition (e.g. cancer). A therapeutic effect
relieves to some extent one or more of the symptoms of the abnormal
condition. In reference to the treatment of a cancer, a therapeutic effect
refers to one or more of the following: (a) a reduction in tumor size; (b)
inhibition (i.e., slowing or stopping) tumor metastasis; (c) inhibition of
tumor growth; and (d) relieving to some extent one or more of the symptoms
associated with the abnormal condition. Compounds demonstrating efficacy
against leukemias can be identified as described herein, except that
rather than inhibiting metastasis, the compounds may instead slow or
decrease cell proliferation or cell growth.
The term "abnormal condition" refers to a function in the cells or tissues
of an organism that deviates from their normal functions in that organism.
An abnormal condition can relate to cell proliferation, cell
differentiation, or cell survival.
Aberrant cell proliferative conditions include cancers such as fibrotic and
mesangial disorders, abnormal angiogenesis and vasculogenesis, wound
healing, psoriasis, diabetes mellitus, and inflammation.
Aberrant differentiation conditions include, but are not limited to
neurodegenerative disorders, slow wound healing rates, and tissue grafting
techniques.
Aberrant cell survival conditions relate to conditions in which programmed
cell death (apoptosis) pathways are activated or abrogated. A number of
protein kinases are associated with the apoptosis pathways. Aberrations in
the function of any one of the protein kinases could lead to cell
immortality or premature cell death.
Cell proliferation, differentiation, and survival are phenomena simply
measured by methods in the art. These methods can involve observing the
number of cells or the appearance of cells under a microscope with respect
to time (for example, days).
The term "administering" relates broadly to the provision to an organism
and more specifically to a method of incorporating a compound into cells
or tissues of an organism. The abnormal condition can be prevented or
treated when the cells or tissues of the organism exist within the
organism or outside of the organism. Cells existing outside the organism
can be maintained or grown in cell culture dishes. For cells harbored
within the organism, many techniques exist in the art to administer
compounds, including (but not limited to) oral, parenteral, dermal,
injection, and aerosol applications. For cells outside of the organism,
multiple techniques exist in the art to administer the compounds,
including (but not limited to) cell microinjection techniques,
transformation techniques, and carrier techniques.
In a preferred embodiment, the invention relates to a method of preventing
or treating an abnormal condition in an organism, where the
azabenzimidazole-based compound has a structure set forth in formula I,
II, or III as defined herein or any of the subgroups thereof set forth
herein.
In another preferred embodiment, the invention relates to a method of
preventing or treating an abnormal condition in an organism, where the
azabenzimidazole compound is selected from the group consisting of SABI
compounds.
In another preferred embodiment, the invention relates to a method of
preventing or treating an abnormal condition in an organism, where the
organism is a mammal.
The term "mammal" refers preferably to such organisms as mice, rats,
rabbits, guinea pigs, and goats, more preferably to monkeys and apes, and
most preferably to humans.
In another preferred embodiment, the invention relates to a method of
preventing or treating an abnormal condition in an organism, where the
abnormal condition is cancer or a fibrotic disorder.
In yet another preferred embodiment, the invention relates to a method of
preventing or treating an abnormal condition in an organism, where the
cancer is selected from the group consisting of lung cancer, ovarian
cancer, breast cancer, brain cancer, intra-axial brain cancer, colon
cancer, prostate cancer, sarcoma, Kaposi's sarcoma, melanoma, and glioma.
In still another preferred embodiment, the invention relates to a method of
preventing or treating an abnormal condition in an organism, where the
method applies to an abnormal condition associated with an aberration in a
signal transduction pathway characterized by an interaction between a
serine/threonine protein kinase and a natural binding partner.
The term "signal transduction pathway" refers to the propagation of a
signal. In general, an extracellular signal is transmitted through the
cell membrane to become an intracellular signal. This signal can then
stimulate a cellular response. The term also encompasses signals that are
propagated entirely within a cell. The polypeptide molecules involved in
signal transduction processes are typically receptor and non-receptor
protein kinases, receptor and non-receptor protein phosphatases,
nucleotide exchange factors, and transcription factors.
The term "aberration", in conjunction with a signal transduction process,
refers to a protein kinase that is over- or under-expressed in an
organism, mutated such that its catalytic activity is lower or higher than
wild-type protein kinase activity, mutated such that it can no longer
interact with a natural binding partner, is no longer modified by another
protein kinase or protein phosphatase, or no longer interacts with a
natural binding partner.
The term "promoting or disrupting the abnormal interaction" refers to a
method that can be accomplished by administering a compound of the
invention to cells or tissues in an organism. A compound can promote an
interaction between a protein kinase and natural binding partners by
forming favorable interactions with multiple atoms at the complex
interface. Alternatively, a compound can inhibit an interaction between a
protein kinase and natural binding partners by compromising favorable
interactions formed between atoms at the complex interface.
In another preferred embodiment, the invention relates to a method of
preventing or treating an abnormal condition in an organism, where the
serine/threonine protein kinase is RAF.
III. Compounds and Pharmaceutical Compositions of the Invention
In another aspect, the invention features azabenzimidazole compounds having
structures set forth in formula I, II, or III:
##STR4##
where (a) R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are independently
selected from the group consisting of
(i) hydrogen;
(ii) saturated or unsaturated alkyl;
(iii) NX.sub.2 X.sub.3, where X.sub.2 and X.sub.3 are independently
selected from the group consisting of hydrogen, saturated or unsaturated
alkyl, and homocylic or heterocyclic ring moieties;
(iv) halogen or trihalomethyl;
(v) a ketone of formula --CO--X.sub.4, where X.sub.4 is selected from the
group consisting of hydrogen, alkyl, and homocylic or heterocyclic ring
moieties;
(vi) a carboxylic acid of formula --(X.sub.5).sub.n --COOH or ester of
formula --(X.sub.6).sub.n --COOO--X.sub.7, where X.sub.5, X.sub.6, and
X.sub.7 are independently selected from the group consisting of alkyl and
homocylic or heterocylic ring moieties and where n is 0 or 1;
(vii) an alcohol of formula (X.sub.8).sub.n --OH or an alkoxy moiety of
formula --(X.sub.8).sub.n --O--X.sub.9, where X.sub.8 and X.sub.9 are
independently selected from the group consisting of hydrogen, saturated or
unsaturated alkyl, and homocyclic or heterocylic ring moieties, where the
ring is optionally substituted with one or more substituents independently
selected from the group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, nitro, and ester and where n is 0 or 1;
(viii) an amide of formula --NHCOX.sub.10, where X.sub.10 is selected from
the group consisting of alkyl, hydroxyl, and homocylic or heterocyclic
ring moieties, where the ring is optionally substituted with one or more
substituents independently selected from the group consisting of alkyl,
alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester;
(ix) a sulfonamide of formula --SO.sub.2 NX.sub.11 X.sub.12, where X.sub.11
and X.sub.12 are selected from the group consisting of hydrogen, alkyl,
and homocyclic or heterocyclic ring moieties;
(x) a homocyclic or heterocyclic ring moiety optionally substituted with
one, two, or three substituents independently selected from the group
consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro,
and ester moieties;
(xi) an aldehyde of formula --CO--H; and
(xii) a sulfone of formula --SO.sub.2 --X.sub.13, where X.sub.13 is
selected from the group consisting of saturated or unsaturated alkyl and
homocylic or heterocyclic ring moieties;
(b) Z.sub.1 and Z.sub.2 are independently selected from the group
consisting of nitrogen, sulfur, oxygen, NH and NR.sub.4, provided that if
one of Z.sub.1 and Z.sub.2 is nitrogen, NH, or NR.sub.4 then the other of
Z.sub.1 and Z.sub.2 is nitrogen, sulfur, oxygen, NH, or NR.sub.4 ; and
(c) Z.sub.3 and X.sub.1 are independently selected from the group
consisting of nitrogen, sulfur, and oxygen.
The term "saturated alkyl" refers to an alkyl moiety that does not contain
any alkene or alkyne moieties. The alkyl moiety may be branched or
non-branched.
The term "unsaturated alkyl" refers to an alkyl moiety that contains at
least one alkene or alkyne moiety. The alkyl moiety may be branched or
non-branched.
The term "amine" refers to a chemical moiety of formula NR.sub.1 R.sub.2
where R.sub.1 and R.sub.2 are independently selected from the group
consisting of hydrogen, saturated or unsaturated alkyl, and homocyclic or
heterocyclic ring moieties, where the ring is optionally substituted with
one or more substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester moieties.
The term "aryl" refers to an aromatic group which has at least one ring
having a conjugated pi electron system and includes both carbocyclic aryl
(e.g. phenyl) and heterocyclic aryl groups (e.g. pyridine). The term
"carbocyclic" refers to a compound which contains one or more covalently
closed ring structures, and that the atoms forming the backbone of the
ring are all carbon atoms. The term thus distinguishes carbocyclic from
heterocyclic rings in which the ring backbone contains at least one atom
which is different from carbon. The term "heteroaryl" refers to an aryl
group which contains at least one heterocyclic ring.
The term "halogen" refers to an atom selected from the group consisting of
fluorine, chlorine, bromine, and iodine.
The term "ketone" refers to a chemical moiety with formula --(R)n--CO--R',
where R and R' are selected from the group consisting of saturated or
unsaturated alkyl and homocyclic or heterocyclic ring moieties and where n
is 0 or 1.
The term "carboxylic acid" refers to a chemical moiety with formula
--(R)n--COOH, where R is selected from the group consisting of saturated
or unsaturated alkyl and homocyclic or heterocyclic ring moieties, and
where n is 0 or 1.
The term "ester" refers to a chemical moiety with formula --(R)n--COOR',
where R and R' are independently selected from the group consisting of
saturated or unsaturated alkyl and homocyclic or heterocylic ring moieties
and where n is 0 or 1.
The term "alcohol" refers to a chemical substituent of formula --ROH, where
R is selected from the group consisting of hydrogen, saturated or
unsaturated alkyl, and homocyclic or heterocylic ring moieties, where the
ring moiety is optionally substituted with one or more substituents
independently selected from the group consisting of alkyl, halogen,
trihalomethyl, carboxylate, nitro, and ester moieties.
The term "amide" refers to chemical substituent of formula --NHCOR, where R
is selected from the group consisting of hydrogen, alkyl, hydroxyl, and
homocylic or heterocylic ring moieties, where the ring is optionally
substituted with one or more substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, or
ester.
The term "alkoxy moiety" refers to a chemical substituent of formula --OR,
where R is hydrogen or a saturated or unsaturated alkyl moiety.
The term "aldehyde" refers to a chemical moiety with formula --(R)n--CHO,
where R is selected from the group consisting of saturated or unsaturated
alkyl and homocyclic or heterocyclic ring moieties and where n is 0 or 1.
The term "sulfone" refers to a chemical moiety with formula --SO.sub.2 --R,
where R is selected from the group consisting of saturated or unsaturated
alkyl and homocyclic or heterocylic ring moieties.
In another preferred embodiment, the invention relates to an
azabenzimidazole-based compound having a structure set forth in formula I,
II, or III, where Z.sub.1 and Z.sub.2 are independently selected from the
group consisting of nitrogen and NH.
In yet another preferred embodiment, the invention relates to an
azabenzimidazole-based compound having a structure set forth in formula I,
II, or III, where R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are independently
selected from the group consisting of hydrogen, saturated or unsaturated
alkyl optionally substituted with a homocyclic or heterocyclic ring
moieties, where the ring moiety is optionally substituted with one, two,
or three substituents independently selected from the group consisting of
alkyl, alkoxy, halogen, trihalomethyl, hydroxy, alkoxy, carboxylate,
nitro, and ester moieties, and a homocyclic or heterocyclic ring moiety
optionally substituted with one, two, or three substituents independently
selected from the group consisting of alkyl, alkoxy, halogen,
trihalomethyl, hydroxy, alkoxy, carboxylate, nitro, and ester moieties.
In other preferred embodiments, the invention relates to an
azabenzimidazole-based compound having a structure set forth in formula I,
II, or III, where R.sub.2 and R.sub.3 are hydrogen.
In another preferred embodiment, the invention relates to an
azabenzimidazole-based compound having a structure set forth in formula I,
II, or III, where R.sub.1 is phenyl optionally substituted with one, two,
or three substituents independently selected from the group consisting of
alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, or ester
moieties.
In yet another preferred embodiment, the invention relates to an
azabenzimidazole-based compound having a structure set forth in formula I,
II, or III, where R.sub.1 selected from the group consisting of SABI
substituents.
The term "SABI substituents" refers to the group of substituents consisting
of phenyl, 2-nitrophenyl, 3-nitrophenyl, 4-nitrophenyl, 2-chlorophenyl,
3-chlorophenyl, 4-chlorophenyl, 2-methylphenyl, 3-methylphenyl,
4-methylphenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl,
2-(trifluoromethyl)phenyl, 3-(trifluoromethyl)phenyl,
4-(trifluoromethyl)phenyl, 2-methoxyphenyl, 3-methoxyphenyl,
4-methoxyphenyl, 2-carboxyphenyl, 3-carboxyphenyl, and 4-carboxyphenyl.
In other preferred embodiments, the invention relates to an
azabenzimidazole-based compound having a structure set forth in formula I,
II, or III, where X.sub.1 is sulfur.
In another preferred embodiment, the invention relates to an
azabenzimidazole-based compound having a structure set forth in formula I,
II, or III, where X.sub.1 is oxygen.
In yet another preferred embodiment, the invention relates to an
azabenzimidazole-based compound having a structure set forth in formula I,
II, or III, where Z.sub.3 is oxygen.
In other preferred embodiments, the invention relates to an
azabenzimidazole-based compound having a structure set forth in formula I,
II, or III, where R.sub.4 is selected from the group consisting of methyl
and ethyl.
In another preferred embodiment, the invention relates to an
azabenzimidazole-based compound having a structure set forth in formula I,
II, or III, where the azabenzimidazole compound is selected from the group
consisting of SABI compounds.
In another aspect, the invention features a pharmaceutical composition
comprising a compound of the invention, as specified herein, or its salt,
and a physiologically acceptable carrier or diluent.
In another aspect, the invention relates to a pharmaceutical composition
comprising a compound having a structure of formula I, II, or II as
defined herein or any of the subgroups thereof set forth herein.
In another preferred embodiment, the invention relates to a pharmaceutical
composition, where the azabenzimidazole compound is selected from the
group consisting of SABI compounds.
The term "pharmaceutical composition" refers to a mixture of an
azabenzimidazole compound of the invention with other chemical components,
such as diluents or carriers. The pharmaceutical composition facilitates
administration of the compound to an organism. Multiple techniques of
administering a compound exist in the art including, but not limited to,
oral, injection, aerosol, parenteral, and topical administration.
Pharmaceutical compositions can also be obtained by reacting compounds
with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric
acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic
acid, p-toluenesulfonic acid, salicyclic acid and the like.
The term "physiologically acceptable" defines a carrier or diluent that
does not abrogate the biological activity and properties of the compound.
The term "carrier" defines a chemical compound that facilitates the
incorporation of a compound into cells or tissues. For example dimethyl
sulfoxide (DMSO) is a commonly utilized carrier as it facilitates the
uptake of many organic compounds into the cells or tissues of an organism.
The term "diluent" defines chemical compounds diluted in water that will
dissolved the compound of interest as well as stabilize the biologically
active form of the compound. Salts dissolved in buffered solutions are
utilized as diluents in the art. One commonly used buffered solution is
phosphate buffered saline because it mimics the salt conditions of human
blood. Since buffer salts can control the pH of a solution at low
concentrations, a buffered diluent rarely modifies the biological activity
of a compound.
IV. Synthetic Methods of the Invention
In another aspect, the invention relates to a method for synthesizing an
azabenzimidazole compound of formula I, II, or III, comprising the steps
of: (a) reacting 2-amino-6-chloro-3-nitropyridine with a second reactant
in a solvent to yield the first intermediate, where the second reactant is
a substituted aryl ring; (b) reducing the first intermediate in the
presence of a catalyst and a reducing agent to yield the second
intermediate; (c) reacting the second intermediate with a third reactant;
and (d) purifying the compound of invention.
In a preferred embodiment, the invention relates to the method of
synthesizing a compound of the invention where the substituted aryl ring
is a substituted phenol, substituted thiophenol, and substituted aniline.
In another preferred embodiment, the invention relates to the method of
synthesizing a compound of the invention where the substituted phenol,
substituted thiophenol, and substituted aniline are selected from the
group consisting of SABI reactants.
The term "SAVI reactants" refers to the group of reactants consisting of
the sodium salt of phenol, 2-nitrophenol, 3-nitrophenol, 4-nitrophenol,
2-chlorophenol, 3-chlorophenol, 4-chlorophenol, 2-cresol, 3-cresol,
4-cresol, 2-fluorophenol, 3-fluorophenol, 4-fluorophenol,
(2-(trifluoromethyl)phenol, (3-(trifluoromethyl)phenol,
(4-(trifluoromethyl)phenol, 2-methoxyphenol, 3-methoxyphenol,
4-methoxyphenol, 2-hydroxybenzoic acid, 3-hydroxybenzoic acid,
4-hydroxybenzoic acid, thiophenol, 2-nitropthiophenol, 3-nitropthiophenol,
4-nitropthiophenol, 2-chlorothiophenol, 3-chlorothiophenol,
4-chlorothiophenol, 2-thiocresol, 3-thiocresol, 4-thiocresol,
2-fluorothiophenol, 3-fluorothiophenol, 4-fluorothiophenol,
(2-(trifluoromethyl)thiophenol, (3-(trifluoromethyl)thiophenol,
(4-(trifluoromethyl)thiophenol, 2-methoxybenzenethiol,
3-methoxybenzenethiol, 4-methoxybenzenethiol, 2-mercaptobenzoic acid,
3-mercaptobenzoic acid, 4-mercaptobenzoic acid, aniline, 2-nitroaniline,
3-nitroaniline, 4-nitroaniline, 2-chloroaniline, 3-chloroaniline,
4-chloroaniline, 2-toluidine, 3-toluidine, 4-toluidine, 2-fluoroaniline,
3-fluoroaniline, 4-fluoroaniline, 2-(trifluoromethyl)aniline,
3-(trifluoromethyl)aniline, 4-(trifluoromethyl)aniline, 2-anisidine,
3-anisidine, 4-anisidine, 2-aminobenzoic acid, 3-aminobenzoic acid, and
4-aminobenzoic acid.
In a preferred embodiment, the invention relates to the method of
synthesizing a compound of the invention where the solvent is n-propanol.
In another preferred embodiment, the invention relates to the method of
synthesizing a compound of the invention where the reducing agent is
hydrogen.
In yet another preferred embodiment, the invention relates to the method of
synthesizing a compound of the invention where the catalyst is Raney
nickel.
In still another preferred embodiment, the invention relates to the method
of synthesizing a compound of the invention where the third reactant is
O-methylisourea.
In another preferred embodiment, the invention relates to the method of
synthesizing a compound of the invention where the third reactant is the
product of the reaction of S-methylisothiouronium sulphate and alkyl
chloroformate.
In yet another preferred embodiment, the invention relates to the method of
synthesizing a compound of the invention where the alkyl chloroformate is
methyl chloroformate.
In still another preferred embodiment, the invention relates to the method
of synthesizing a compound of the invention where the alkyl chloroformate
is ethyl chloroformate.
The summary of the invention described above is non-limiting and other
features and advantages of the invention will be apparent from the
following description of the preferred embodiments, and from the claims.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is directed in part towards methods of modulating the
function of serine/threonine protein kinases with azabenzimidazole-based
compounds. In addition, the invention relates in part to methods for
identifying compounds that modulate the function of serine/threonine
protein kinases. The methods incorporate cells that express a
serine/threonine protein kinase, such as RAF.
RAF is a non-receptor protein kinase that is recruited to the cell membrane
when it binds to activated RAS, a guanine triphosophate hydrolyzing
enzyme. RAS is activated when an activated receptor protein tyrosine
kinase, such as EGFR or PDGFR, bind to an adaptor protein, GRB2, and a
guanine nucleotide exchange factor, SOS. SOS removes guanine diphosphate
from RAS, replaces it with guanine triphosphate, and thereby activates
RAS. RAS then binds RAF and consequently activates RAF. RAF may then
phosphorylate other protein targets on serine and threonine residues, such
as the kinase (MEK) that phosphorylates and consequently activates
mitogen-activated protein kinase (MAPK). Thus, RAF serves as an
intermediary controlling factor in mitogen-activated signal transduction.
Due to the important regulatory role of RAF in cells, modifications to the
amino acid sequence of RAF can alter its function and consequently modify
cellular behavior. RAF's role in cell proliferation is underscored by the
observation that mutations to RAF's amino acid sequence have been
associated with tumors and cancers. Because the mutations to RAF that give
rise to cancer in cells lead to RAF molecules that display unregulated
catalytic activity, inhibitors of RAF may alleviate or even abrogate the
cell proliferation that leads to cancer in these cells.
Methods of the present invention can detect compounds that modulate the
function of the protein kinase RAF in cells. RAF phosphorylates a protein
kinase (MEK) which in turn phosphorylates mitogen-activated protein kinase
(MAPK). Assays that monitor only the phosphorylation of MEK by RAF are not
sensitive because the phosphorylation levels of MEK are not significant.
To overcome this sensitivity dilemma, the phosphorylation of both MEK and
MAPK are followed in the assays of the present invention. The MAPK
phosphorylation signal amplifies the MEK phosphorylation signal and allows
RAF-dependent phosphorylation to be followed in enzyme-linked
immunosorbant assays. In addition, the assay of the invention is
preferably performed in a high throughput format such that many compounds
can be rapidly monitored in a short period of time.
The methods of the present invention have identified compounds that inhibit
the function of the RAF protein kinase. These compounds are
azabenzimidazole-based derivatives. Although azabenzimidazole-based
derivatives have been tested for their ability to inhibit enzymes involved
with nucleotide synthesis in bacteria, many of these compounds have not
yet been significantly explored with respect to protein kinase inhibition.
Because RAF exhibits significant amino acid homology to other
serine/threonine protein kinases, the azabenzimidazole-based compounds of
the invention may likely inhibit serine/threonine protein kinases other
than RAF. Thus, the methods of the invention also relate to
serine/threonine protein kinases other than RAF, including receptor and
non-receptor serine/threonine protein kinases.
The methods of the invention also pertain to other compounds that modulate
RAF function in cells as the high throughput aspect of the methods allows
a wide array of molecules to be tested in short period of time. Therefore,
the methods of the invention can identify existing molecules not disclosed
in the present invention that modulate STK function.
I. Biological Activity of Azabenzimidazole-Based Compounds
Azabenzimidazole-based compounds of the present invention were tested for
their ability to inhibit RAF protein kinase function. The biological
assays and results of these inhibition studies are reported herein. The
methods used to measure azabenzimidazole-based compound modulation of
protein kinase function are similar to those described in U.S. application
Ser. No. 08/702,232 now abandoned, by Tang et al., and entitled
"Indolinone Combinatorial Libraries and Related Products and Methods for
the Treatment of Disease," filed Aug. 23, 1996, with respect to the high
throughput aspect of the method. The Ser. No. 08/702,232 application is
incorporated herein by reference in its entirety, including any drawings.
II. Target Diseases to be Treated by Azabenzimidazole-Based Compounds
The methods, compounds, and pharmaceutical compositions described herein
are designed to inhibit cell proliferative disorders by modulating the
function of the RAF protein kinase. Proliferative disorders result in
unwanted cell proliferation of one or more subsets of cells in a
multicellular organism resulting in harm to the organism. The methods,
compounds, and pharmaceutical compositions described herein may also be
useful for treating and preventing other disorders in organisms, such as
disorders related to premature cell death (i.e., neurological diseases) or
inflammation. These disorders may be a result of RAF molecules that
function inappropriately or a result of RAF-related protein kinase
molecules that function inappropriately.
Alterations in the function of the RAF protein kinase or protein kinases
related to RAF can lead to enhanced or decreased cell proliferative
conditions evident in certain diseases. Aberrant cell proliferative
conditions include cancers, fibrotic disorders, mesangial disorders,
abnormal angiogenesis and vasculogenesis, wound healing, psoriasis,
restenosis, and inflammation.
Fibrotic disorders relate to the abnormal formation of the cellular
extracellular matrix. An example of a fibrotic disorder is hepatic
cirrhosis. Hepatic cirrhosis is characterized by an increased
concentration of extracellular matrix constituents resulting in the
formation of a hepatic scar. Hepatic cirrhosis can cause diseases such as
cirrhosis of the liver.
Mesangial cell proliferative disorders occur due to the abnormal
proliferation of mesangial cells. Mesangial proliferative disorders
include various human renal diseases, such as glomerulonephritis, diabetic
nephropathy, malignant nephrosclerosis, thrombotic microangiopathy
syndromes, transplant rejection, and glomerulopathies.
Preferred types of cancers that may be treated by the methods and compounds
of the invention are lung cancer, ovarian cancer, breast cancer, brain
cancer, intra-axial brain cancer, colon cancer, prostate, Kaposi's
sarcoma, melanoma, and glioma. Evidence that the compounds and methods of
the invention can effectively be utilized to stem and reverse the
proliferation of cancer cells is provided herein by reference.
Angiogenic and vasculogenic disorders result from excess proliferation of
blood vessels. Blood vessel proliferation is necessary in a variety of
normal physiological processes such as embryonic development, corpus
luteum formation, wound healing and organ regeneration. However, blood
vessel proliferation is also essential in cancer tumor development. Other
examples of blood vessel proliferative disorders include arthritis, where
new capillary blood vessels invade the joint and destroy cartilage. In
addition, blood vessel proliferative diseases include ocular diseases,
such as diabetic retinopathy, where new capillaries in the retina invade
the vitreous, bleed and cause blindness. Conversely, disorders related to
the shrinkage, contraction or closing of blood vessels, such as
restenosis, are also implicated in adverse regulation of protein kinases.
Moreover, vasculogenesis and angiogenesis are associated with the growth of
malignant solid tumors and metastasis. A vigorously growing cancer tumor
requires a nutrient and oxygen rich blood supply to continue growing. As a
consequence, an abnormally large number of capillary blood vessels often
grow in concert with the tumor and act as supply lines to the tumor. In
addition to supplying nutrients to the tumor, the new blood vessels
embedded in a tumor provide a gateway for tumor cells to enter the
circulation and metastasize to distant sites in the organism. Folkman,
1990, J. Natl. Cancer Inst. 82:4-6.
Inappropriate RAF activity can stimulate cell proliferative disorders.
Molecules specifically designed to modulate the function of the RAF
protein kinase have been shown to inhibit cellular proliferation.
Specifically, antisense nucleic acid molecules, which are designed to both
bind to message RNA encoding the RAF protein kinase and block translation
from that message, effectively reversed transformation of A549 cells in
vitro. Monia et al., 1996, Nature Medicine 2: 688, incorporated herein by
reference in its entirety including all figures and tables. A549 cells are
human malignant cells.
These RAF-targeted antisense studies provide evidence that the
azabenzimidazole molecules of the invention, which modulate the function
of the RAF protein kinase, can stem, and likely reverse, the proliferation
of malignant cells in an organism. These azabenzimidazole compounds can be
tested in the in vitro methods provided herein by example. Furthermore,
the azabenzimidazole compounds may be tested for their effect upon tumor
cells in vivo by the xenograft methods also provided herein by example.
There exist at least two ways in which inappropriate RAF activity can
stimulate unwanted cell proliferation of a particular type of cells: (1)
directly stimulating growth of the particular cell, or (2) increasing
vascularization of a particular area, such as tumor tissue, thereby
facilitating growth of the tissue.
The use of the present invention is facilitated by first identifying
whether the cell proliferation disorder is RAF driven. Once such disorders
are identified, patients suffering from such a disorder can be identified
by analysis of their symptoms using procedures well known to physicians or
veterinarians of ordinary skill in the art. Such patients can then be
treated as described herein.
Determining whether the cell proliferation disorder is RAF driven may be
accomplished by first determining the level of RAF activity occurring in
the cell or in a particular location in a patient's body. For example, in
the case of cancer cells the level of one or more RAF activities may be
compared for non-RAF driven cancers and RAF driven cancels. If the cancer
cells have a higher level of RAF activity than RAF driven cancers,
preferably equal to or greater than RAF driven cancers, then they are
candidates for treatment using the described RAF-modulating methods and
compounds of the invention.
In the case of cell proliferative disorders arising due to unwanted
proliferation of non-cancer cells, the level of RAF activity is compared
to that level occurring in the general population (e.g., the average level
occurring in the general population of people or animals excluding those
people or animals suffering from a cell proliferative disorder). If the
unwanted cell proliferation disorder is characterized by a higher RAF
level than occurring in the general population then the disorder is a
candidate for treatment using the described RAF modulating methods and
compounds of the invention.
III. Pharmaceutical Compositions and Administration of
Azabenzimidazole-Based Compounds
Methods of preparing pharmaceutical formulations of the compounds, methods
of determining the amounts of compounds to be administered to a patient,
and modes of administering compounds to an organism are disclosed in U.S.
application Ser. No. 08/702,232, by Tang et. al., and entitled "Indolinone
Combinatorial Libraries and Related Products and Methods for the Treatment
of Disease," filed Aug. 23, 1996, and International patent publication
number WO 96/22976, by Buzzetti et al., and entitled "Hydrosoluble
3-Arylidine-2-Oxindole Derivatives as Tyrosine Kinase Inhibitors,"
published Aug. 1, 1996, both of which are incorporated herein by reference
in their entirety, including any drawings. Those skilled in the art will
appreciate that such descriptions are applicable to the present invention
and can be easily adapted to it.
EXAMPLES
The examples below are non-limiting and are merely representative of
various aspects and features of the present invention. The examples
describe methods for synthesizing compounds of the invention and methods
for measuring an effect of a compound on the function of the RAF protein
kinase.
The cells used in the methods are commercially available. The nucleic acid
vectors harbored by the cells are also commercially available and the
sequences of genes for the various protein kinases are readily accessible
in sequence data banks. Thus, a person of ordinary skill in the art can
readily recreate the cell lines in a timely manner by combining the
commercially available cells, the commercially available nucleic acid
vectors, and the protein kinase genes using techniques readily available
to persons of ordinary skill in the art.
Example 1
Procedures for Synthesizing Azabenzimidazole Compounds of the Invention
The invention will now be illustrated in the following non-limiting
examples in which, unless otherwise stated:
(i) evaporations were carried out by rotary evaporation under vacuum;
(ii) operations were carried out under an atmosphere of an inert gas such
as nitrogen;
(iii) high performance liquid chromatography (HPLC) was performed on Merck
LiChrosorb RP-18 reversed-phase silica obtained from E. Merck, Darmstadt,
Germany;
(iv) yields are given for illustration only and are not necessarily the
maximum attainable;
(v) melting points are uncorrected and were determined using a HWS Mainz SG
2000 digital melting point apparatus;
(vi) the structures of all compounds of the formula I, II, and III of this
invention were confirmed by proton magnetic resonance spectroscopy on a
Bruker AMX500-NMR spectrophotometer, by elemental microanalysis and, in
certain cases, by mass spectroscopy;
(vii) the purity of the structures were performed by thin layer
chromatography (TLC) using silica gel (Merck Silica Gel 60 F254) or by
HPLC; and
(vii) intermediates were not generally fully characterized and purity was
assessed by thin layer chromatography (TLC) or by HPLC.
SYNTHETIC PROCEDURES
Compound A-90:
2-Methoxycarbonylamino-(6-phenylmercapto-3H-imidazol[4,5-b]pyridine
2-amino-3-nitro-6-(phenylmercapto)pyridine was prepared by heating
2-amino-6-chloro-3-nitropyridine (84.0 g, 0.484 mol) and sodium
thiophenolate (Fluka) (72.0 g, 0.545 mol) in 2-propanol (1500 mL) under
reflux for 2 hours. After cooling to room temperature the suspension was
diluted with water (100 ml), the solid was collected by vacuum filtration,
washed with water and 2-propanol, and dried at 50.degree. C. under vacuum
to give 109.1 g (95% yield) of 2-amino-3-nitro-6-(phenylmercapto)pyridine,
m.p. 148-152.degree. C.).
2,3-Diamino-6-(phenylmercapto)pyridine was prepared by hydrogenating
2-amino-3-nitro-6-(phenylmercapto)pyridine (107.1 g, 0.433 mol) under 5
atm of H.sub.2 in the presence of 30 g of Raney-Ni in 1200 mL of
2-propanol at 70.degree. C. After 4 hours (29.1 L of hydrogen) the
reaction mixture was cooled to 4.degree. C. while stirring continuously.
The precipitate was collected by vacuum filtration, washed with 2-propanol
and dried at 50.degree. C. under vacuum. The combined filtrates were
concentrated under reduced pressure and recrystallized from 2-propanol.
After washing the hydrogenation apparatus twice with 1000 mL of THF,
evaporation under reduced pressure and recrystallization from 2-propanol,
the precipitate was collected and dried at 50.degree. C. under vacuum to
give 80.4 g (87.1% yield) of 2,3-diamino-6-(phenylmercapto)pyridine m.p.
119-122.degree. C.)
2-Methoxycarbonylamino-6-phenylmercapto-3H-imidazo[4,5-b]pyridine was
prepared by adding methyl chloroformate (34 mL, 0.44 mol) dropwise to a
cold (5-15.degree. C.) solution of S-methylisothiouronium sulphate (53 g,
0.19 mol) (Aldrich) in 68 mL of water while the temperature was kept below
20.degree. C. Afterwards, aqueous sodium hydroxide (116 g, 25% NaOH) was
added carefully and a white precipitate occurred. After 20 minutes, water
(210 mL) was added and the pH adjusted to 4.0 with acetic acid (glacial,
34 mL). To this mixture a solution of
2,3-diamino-6-(phenylmercapto)pyridine (37.8 g, 0.174 mol) in 210 mL of
ethanol was added dropwise and heated to 85-90.degree. C. for 2 hours.
After cooling the reaction mixture overnight, the precipitate was isolated
by filtration, washed with warm water (1000 mL), dried and recrystallized
from acetic acid and ethanol at 4.degree. C. The precipitate was collected
by filtration, washed with methanol and dried at 50.degree. C. under
vacuum to give 30 g (57.4% yield) of
2-methoxycarbonylamino-6-phenylmercapto-3H-imidazo[4,5-b]pyridine, m.p.
269-274.degree. C. (dec.).
Compound A-3: 2-Methoxycarbonylamino-6-phenoxy-3H-imidazo[4,5-b]pyridine
By substituting sodium phenolate in place of sodium thiophenolate in
Example A-90, the identical process gives
2-methoxycarbonylamino-6-phenoxy-3H-imidazo[4,5-b]pyridine, m.p.
>280.degree. C. (dec.).
Compound A-4: 2-Ethoxycarbonylamino-6-phenoxy-3H-imidazo[4,5-b]pyridine
By substituting ethyl chloroformate in place of methyl chloroformate in
Example A-3, the identical process gives
2-ethoxycarbonylamino-6-phenoxy-3H-imidazo[4,5-b]pyridine, m.p.
>280.degree. C. (dec.).
Compound A-1: 2-Oxo-6-phenoxy-3H-imidazol[4,5-b]pyridine
By reacting O-methylisourea directly with
2,3-diamino-6-(phenylmercapto)pyridine in place of the product of the
reaction of S-methylisothiouronium sulfate and methyl chloroformate in
Example A-3, the identical process gives
2-oxo-6-phenoxy-3H-imidazol[4,5-b]pyridine, m.p. 277-278.degree. C.
Compound A-2: 2-Oxo-6-phenylmercapto-3H-imidazol[4,5-b]pyridine
By substituting sodium thiophenolate in place of sodium phenolate in
Example A-1, the identical process gives
2-oxo-6-phenylmercapto-3H-imidazol[4,5-b]pyridine, m.p. 253-254.degree. C.
Compounds A-5-A-25:
By substituting the appropriate phenolate for sodium phenolate in Example
A-1, the identical process gives the following examples. For examples
A-23, A-24, and A-25, the carboxy groups are protected by a methyl, ethyl,
benzyl, t-butyl or other suitable ester and then deprotected in the last
step to give the compounds.
______________________________________
A-5 2-Oxo-6-(2-nitrophenoxy)-3H-imidazo[4,5-
b]pyridine
A-6 2-Oxo-6-(3-nitrophenoxy)-3H-imidazo[4,5-
b]pyridine
A-7 2-Oxo-6-(4-nitrophenoxy)-3H-imidazo[4,5-
b]pyridine
A-8 2-Oxo-6-(2-chlorophenoxy)-3H-imidazo[4,5-
b]pyridine
A-9 2-Oxo-6-(3-chlorophenoxy)-3H-imidazo[4,5-
b]pyridine
A-10 2-Oxo-6-(4-chlorophenoxy)-3H-imidazo[4,5-
b]pyridine
A-11 2-Oxo-6-(2-methylphenoxy)-3H-imidazo[4,5-
b]pyridine
A-12 2-Oxo-6-(3-methylphenoxy)-3H-imidazo[4,5-
b]pyridine
A-13 2-Oxo-6-(4-methylphenoxy)-3H-imidazo[4,5-
b]pyridine
A-14 2-Oxo-6-(2-fluorophenoxy)-3H-imidazo[4,5-
b]pyridine
A-15 2-Oxo-6-(3-fluorophenoxy)-3H-imidazo[4,5-
b]pyridine
A-16 2-Oxo-6-(4-fluorophenoxy)-3H-imidazo[4,5-
b]pyridine
A-17 2-Oxo-6-[2-(trifluoromethyl)phenoxy]-3H-
imidazo[4,5-b]pyridine
A-18 2-Oxo-6-[3-(trifluoromethyl)phenoxy]-3H-
imidazo[4,5-b]pyridine
A-19 2-Oxo-6-[4-(trifluoromethyl)phenoxy]-3H-
imidazo[4,5-b]pyridine
A-20 2-Oxo-6-(2-methoxyphenoxy)-3H-imidazo[4,5-
b]pyridine
A-21 2-Oxo-6-(3-methoxyphenoxy)-3H-imidazo[4,5-
b]pyridine
A-22 2-Oxo-6-(4-methoxyphenoxy)-3H-imidazo[4,5-
b]pyridine
A-23 2-Oxo-6-(2-carboxyphenoxy)-3H-imidazo[4,5-
b]pyridine
A-24 2-Oxo-6-(3-carboxyphenoxy)-3H-imidazo[4,5-
b]pyridine
A-25 2-Oxo-6-(4-carboxyphenoxy)-3H-imidazo[4,5-
b]pyridine
______________________________________
Compounds A-26-A-46:
By substituting the appropriate thiophenolate for sodium thiophenolate in
Example A-2, the identical process gives the following examples. For
examples A-44, A-45, and A-46, the carboxy groups are protected by a
methyl, ethyl, benzyl, t-butyl or other suitable ester and then
deprotected in the last step to give the compounds.
______________________________________
A-26 2-Oxo-6-(2-nitrophenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-27 2-Oxo-6-(3-nitrophenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-28 2-Oxo-6-(4-nitrophenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-29 2-Oxo-6-(2-chlorophenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-30 2-Oxo-6-(3-chlorophenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-31 2-Oxo-6-(4-chlorophenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-32 2-Oxo-6-(2-methylphenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-33 2-Oxo-6-(3-methylphenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-34 2-Oxo-6-(4-methylphenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-35 2-Oxo-6-(2-fluorophenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-36 2-Oxo-6-(3-fluorophenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-37 2-Oxo-6-(4-fluorophenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-38 2-Oxo-6-[2-(trifluoromethyl)phenylmercapto]-3H-
imidazo[4,5-b]pyridine
A-39 2-Oxo-6-[3-(trifluoromethyl)phenylmercapto]-3H-
imidazo[4,5-b]pyridine
A-40 2-Oxo-6-[4-(trifluoromethyl)phenylmercapto]-3H-
imidazo[4,5-bipyridine
A-41 2-Oxo-6-(2-methoxyphenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-42 2-Oxo-6-(3-methoxyphenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-43 2-Oxo-6-(4-methoxyphenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-44 2-Oxo-6-(2-carboxyphenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-45 2-Oxo-6-(3-carboxyphenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-46 2-Oxo-6-(4-carboxyphenylmercapto)-3H-imidazo[4,5-
b]pyridine
______________________________________
Compounds A-47-A-68:
By substituting the appropriate aniline salt for sodium phenolate in
Example A-1, the identical process gives the following examples. For
examples A-66, A-67, and A-68, the carboxy groups are protected by a
methyl, ethyl, benzyl, t-butyl or other suitable ester and then
deprotected in the last step to give the compounds.
______________________________________
A-47 2-Oxo-6-phenylamino-3H-imidazo[4,5-b]pyridine
A-48 2-Oxo-6-(2-nitrophenylamino)-3H-imidazo[4,5-
b]pyridine
A-49 2-Oxo-6-(3-nitrophenylamino)-3H-imidazo[4,5-
b]pyridine
A-50 2-Oxo-6-(4-nitrophenylamino)-3H-imidazo[4,5-
b]pyridine
A-51 2-Oxo-6-(2-chlorophenylamino)-3H-imidazo[4,5-
b]pyridine
A-52 2-Oxo-6-(3-chlorophenylamino)-3H-imidazo[4,5-
b]pyridine
A-53 2-Oxo-6-(4-chlorophenylamino)-3H-imidazo[4,5-
b]pyridine
A-54 2-Oxo-6-(2-methylphenylamino)-3H-imidazo[4,5-
b]pyridine
A-55 2-Oxo-6-(3-methylphenylamino)-3H-imidazo[4,5-
b]pyridine
A-56 2-Oxo-6-(4-methylphenylamino)-3H-imidazo[4,5-
b]pyridine
A-57 2-Oxo-6-(2-fluorophenylamino)-3H-imidazo[4,5-
b]pyridine
A-58 2-Oxo-6-(3-fluorophenylamino)-3H-imidazo[4,5-
b]pyridine
A-59 2-Oxo-6-(4-fluorophenylamino)-3H-imidazo[4,5-
b]pyridine
A-60 2-Oxo-6-[2-(trifluoromethyl)phenylamino[-3H-
imidazo[4,5-b]pyridine
A-61 2-Oxo-6-[3-(trifluoromethyl)phenylamino]-3H-
imidazo[4,5-b]pyridine
A-62 2-Oxo-6-[4-(trifluoromethyl)phenylamino]-3H-
imidazo[4,5-b]pyridine
A-63 2-Oxo-6-(2-methoxyphenylamino)-3H-imidazo[4,5-
b]pyridine
A-64 2-Oxo-6-(3-methoxyphenylamino)-3H-imidazo[4,5-
b]pyridine
A-65 2-Oxo-6-(4-methoxyphenylamino)-3H-imidazo[4,5-
b]pyridine
A-66 2-Oxo-6-(2-carboxyphenylamino)-3H-imidazo[4,5-
b]pyridine
A-67 2-Oxo-6-(3-carboxyphenylamino)-3H-imidazo[4,5-
b]pyridine
A-68 2-Oxo-6-(4-carboxyphenylamino)-3H-imidazo[4,5-
b]pyridine
______________________________________
Compounds A-69-A-89:
By substituting the appropriate phenolate for sodium phenolate in Example
A-3, the identical process gives the following examples. For examples
A-87, A-88, and A-89, the carboxy groups are protected by a methyl, ethyl,
benzyl, t-butyl or other suitable ester and then deprotected in the last
step to give the compounds.
______________________________________
A-69 2-Methoxycarbonylamino-6 (2-nitrophenoxy)-3H-
imidazo[4,5-b]pyridine
A-70 2-Methoxycarbonylamino-6-(3-nitrophenoxy)-3H-
imidazo[4,5-b]pyridine
A-71 2-Methoxycarbonylamino-6-(4-nitrophenoxy)-3H-
imidazo[4,5-b]pyridine
A-72 2-Methoxycarbonylamino-6-(2-chlorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-73 2-Methoxycarbonylamino-6-(3-chlorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-74 2-Methoxycarbonylamino-6-(4-chlorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-75 2-Methoxycarbonylamino-6-(2-methylphenoxy)-3H-
imidazo[4,5-b]pyridine
A-76 2-Methoxycarbonylamino-6-(3-methylphenoxy)-3H-
imidazo[4,5-b]pyridine
A-77 2-Methoxycarbonylamino-6-(4-methylphenoxy)-3H-
imidazo[4,5-b]pyridine
A-78 2-Methoxycarbonylamino-6-(2-fluorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-79 2-Methoxycarbonylamino-6-(3-fluorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-80 2-Methoxycarbonylamino-6-(4-fluorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-81 2-Methoxycarbonylamino-6-[2-
(trifluoromethyl)phenoxy]-3H-imidazo[4,5-
b]pyridine
A-82 2-Methoxycarbonylamino-6-[3-
(trifluoromethyl)phenoxy]-3H-imidazo[4,5-
b]pyridine
A-83 2-Methoxycarbonylamino-6-[4-
(trifluoromethyl)phenoxy]-3H-imidazo[4,5-
b]pyridine
A-84 2-Methoxycarbonylamino-6-(2-methoxyphenoxy)-3H-
imidazo[4,5-b]pyridine
A-85 2-Methoxycarbonylamino-6-(3-methoxyphenoxy)-3H-
imidazo[4,5-b]pyridine
A-86 2-Methoxycarbonylamino-6-(4-methoxyphenoxy)-3H-
imidazo[4,5-b]pyridine
A-87 2-Methoxycarbonylamino-6-(2-carboxyphenoxy)-3H-
imidazo[4,5-b]pyridine
A-88 2-Methoxycarbonylamino-6-(3-carboxyphenoxy)-3H-
imidazo[4,5-b]pyridine
A-89 2-Methoxycarbonylamino-6-(4-carboxyphenoxy)-3H-
imidazo[4,5-b]pyridine
______________________________________
Compounds A-91-A-111:
By substituting the appropriate thiophenolate for sodium thiophenolate in
Example A-90, the identical process gives the following examples. For
examples A-109, A-110, and A-111, the carboxy groups are protected by a
methyl, ethyl, benzyl, t-butyl or other suitable ester and then
deprotected in the last step to give the compounds.
______________________________________
A-91 2-Methoxycarbonylamino-6-(2-nitrophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-92 2-Methoxycarbonylamino-6-(3-nitrophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-93 2-Methoxycarbonylamino-6-(4-nitrophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-94 2-Methoxycarbonylamino-6-(2-
chlorophenylmercapto)-3H-imidazo[4,5-b]pyridine
A-95 2-Methoxycarbonylamino-6-(3-
chlorophenylmercapto)-3H-imidazo[4,5-b]pyridine
A-96 2-Methoxycarbonylamino-6-(4-
chlorophenylmercapto)-3H-imidazo[4,5-b]pyridine
A-97 2-Methoxycarbonylamino-6-(2-
methylphenylmercapto-3H-imidazo[4,5-b]pyridine
A-98 2-Methoxycarbonylamino-6-(3-
methylphenylmercapto)-3H-imidazo[4,5-b]pyridine
A-99 2-Methoxycarbonylamino-6-(4-
methylphenylmercapto)-3H-imidazo[4,5-b]pyridine
A-100 2-Methoxycarbonylamino-6-(2-
fluorophenylmercapto)-3H-imidazo[4,5-b]pyridine
A-101 2-Methoxycarbonylamino-6-(3-
fluorophenylmercapto)-3H-imidazo[4,5-b]pyridine
A-102 2-Methoxycarbonylamino-6-(4-
fluorophenylmercapto)-3H-imidazo[4,5-b]pyridine
A-103 2-Methoxycarbonylamino-6-[2-
(trifluoromethyl)phenylmercapto]-3H-imidazo[4,5-
b]pyri dine
A-104 2-Methoxycarbonylamino-6-[3-
(trifluoromethyl)phenylmercapto-3H-imidazo[4,5-
b]pyri dine
A-105 2-Methoxycarbonylamino-6-[4-
(trifluoromethyl)phenylmercapto]-3H-imidazo[4,5-
b]pyri dine
A-106 2-Methoxycarbonylamino-6-(2-
methoxyphenylmercapto)-3H-imidazo[4,5-b]pyridine
A-107 2-Methoxycarbonylamino-6-(3-
methoxyphenylmercapto)-3H-imidazo[4,5-b]pyridine
A-108 2-Methoxycarbonylamino-6-(4-
methoxyphenylmercapto)-3H-imidazo[4,5-b]pyridine
A-109 2-Methoxycarbonylamino-6-(2-
carboxyphenylmercapto)-3H-imidazo[4,5-b]pyridine
A-110 2-Methoxycarbonylamino-6-(3-
carboxyphenylmercapto)-3H-imidazo[4,5-b]pyridine
A-111 2-Methoxycarbonylamino-6-(4-
carboxyphenylmercapto)-3H-imidazo[4,5-b]pyridine
______________________________________
Compounds A-112-A-133:
By substituting the appropriate aniline salt for sodium phenolate in
Example A-3, the identical process gives the following examples. For
examples A-131, A-132, and A-133, the carboxy groups are protected by a
methyl, ethyl, benzyl, t-butyl or other suitable ester and then
deprotected in the last step to give the compounds.
______________________________________
A-112 2-Methoxycarbonylamino-6-phenylamino-3H-
imidazo[4,5-A-112
A-113 2-Methoxycarbonylamino-6-(2-nitrophenylamino)-3H-
imidazo[4,5-b]pyridine
A-114 2-Methoxycarbonylamino-6-(3-nitrophenylamino)-3H-
imidazo[4,5-b]pyridine
A-115 2-Methoxycarbonylamino-6-(4-nitrophenylamino)-3H-
imidazo[4,5-b]pyridine
A-116 2-Methoxycarbonylamino-6-(2-chlorophenylamino)-
3H-imidazo[4,5-b]pyridine
A-117 2-Methoxycarbonylamino-6-(3-chlorophenylamino)-
3H-imidazo[4,5-b]pyridine
A-118 2-Methoxycarbonylamino-6-(4-chlorophenylamino)-
3H-imidazo[4,5-b]pyridine
A-119 2-Methoxycarbonylamino-6-(2-methylphenylamino)-
3H-imidazo[4,5-b]pyridine
A-120 2-Methoxycarbonylamino-6-(3-methylphenylamino)-
3H-imidazo[4,5-b]pyridine
A-121 2-Methoxycarbonylamino-6-(4-methylphenylamino)-
3H-imidazo[4,5-b]pyridine
A-122 2-Methoxycarbonylamino-6-(2-fluorophenylamino)-
3H-imidazo[4,5-b]pyridine
A-123 2-Methoxycarbonylamino-6-(3-fluorophenylamino)-
3H-imidazo[4,5-b]pyridine
A-124 2-Methoxycarbonylamino-6-(4-fluorophenylamino)
3H-imidazo[4,5-b]pyridine
A-125 2-Methoxycarbonylamino-6-[2-
(trifluoromethyl)phenylamino]-3H-imidazo[4,5-
b]pyridine
A-126 2-Methoxycarbonylamino-6-[3-
(trifluoromethyl) phenylamino]-3H-imidazo[4,5-
b]pyridine
A-127 2-Methoxycarbonylamino-6-[4-
(trifluoromethyl)phenylamino]-3H-imidazo[4,5-
b]pyridine
A-128 2-Methoxycarbonylamino-6-(2-methoxyphenylamino)-
3H-imidazo[4,5-b]pyridine
A-129 2-Methoxycarbonylamino-6-(3-methoxyphenylamino)-
3H-imidazo[4,5-b]pyridine
A-130 2-Methoxycarbonylamino-6-(4-methoxyphenylamino)-
3H-imidazo[4,5-b]pyridine
A-131 2-Methoxycarbonylamino-6-(2-carboxyphenylamino)-
3H-imidazo[4,5-b]pyridine
A-132 2-Methoxycarbonylamino-6-(3-carboxyphenylamino)-
3H-imidazo[4,5-b]pyridine
A-133 2-Methoxycarbonylamino-6-(4-carboxyphenylamino)-
3H-imidazo[4,5-b]pyridine
______________________________________
Compounds A-134-A-154:
By substituting the appropriate phenolate for sodium phenolate in Example
A-4, the identical process gives the following examples. For examples
A-152, A-153, and A-154, the carboxy groups are protected by a methyl,
ethyl, benzyl, t-butyl ester or other suitable ester and then deprotected
in the last step to give the compounds.
______________________________________
A-134 2-Ethoxycarbonylamino-6-(2-nitrophenoxy)-3H-
imidazo[4,5-b]pyridine
A-135 2-Ethoxycarbonylamino-6-(3-nitrophenoxy)-3H-
imidazdo[4,5-b]pyridine
A-136 2-Ethoxycarbonylamino-6-(4-nitrophenoxy)-3H-
imidazo[4,5-b]pyridine
A-137 2-Ethoxycarbonylamino-6-(2-chlorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-138 2-Ethoxycarbonylamino-6 (3-chlorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-139 2-Ethoxycarbonylamino-6-(4-chlorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-140 2-Ethoxycarbonylamino-6-(2-methylphenoxy)-3H-
imidazo[4,5-b]pyridine
A-141 2-Ethoxycarbonylamino-6-(3-methylphenoxy)-3H-
imidazo[4,5-b]pyridine
A-142 2-Ethoxycarbonylamino-6-(4-methylphenoxy)-3H-
imidazo[4,5-b]pyridine
A-143 2-Ethoxycarbonylamino-6-(2-fluorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-144 2-Ethoxycarbonylamino-6-(3-fluorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-145 2-Ethoxycarbonylamino-6-(4-fluorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-146 2-Ethoxycarbonylamino-6-[2-
(trifluoromethyl)phenoxy]-3H-imidazo[4,5-
b]pyridine
A-147 2-Ethoxycarbonylamino-6-[3-
(trifluoromethyl)phenoxy]-3H-imidazo[4,5-
b]pyridine
A-148 2-Ethoxycarbonylamino-6-[4-
(trifluoromethyl)phenoxy]-3H-imidazo[4,5-
b]pyridine
A-149 2-Ethoxycarbonylamino-6-(2-methoxyphenoxy)-3H-
imidazo[4,5-b]pyridine
A-150 2-Ethoxycarbonylamino-6-(3-methoxyphenoxy)-3H-
imidazo[4,5-b]pyridine
A-151 2-Ethoxycarbonylamino-6-(4-methoxyphenoxy)-3H-
imidazo[4,5-b]pyridine
A-152 2-Ethoxycarbonylamino-6-(2-carboxyphenoxy)-3H-
imidazo[4,5-b]pyridine
A-153 2-Ethoxycarbonylatrino-6-(3-carboxyphenoxy)-3H-
imidazo[4,5-b]pyridine
A-154 2-Ethoxycarbonylamino-6-(4-carboxyphenoxy)-3H-
imidazo[4,5-b]pyridine
______________________________________
Compounds A-155-A-176:
By substituting the appropriate thiophenolate for sodium phenolate in
Example A-4, the identical process gives the following examples. For
examples A-174, A-175, and A-176, the carboxy groups are protected by a
methyl, ethyl, benzyl, t-butyl or other suitable ester and then
deprotected in the last step to give the compounds.
______________________________________
A-155 2-Ethoxycarbonylamino-6-phenylmercapto-3H-
imidazo[4,5-b]pyridine
A-156 2-Ethoxycarbonylamino-6-(2-nitrophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-157 2-Ethoxycarbonylamino-6-(3-nitrophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-158 2-Ethoxycarbonylamino-6-(4-nitrophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-159 2 -Ethoxycarbonylamino-6-(2-chlorophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-160 2 Ethoxycarbonylamino-6-(3-chlorophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-161 2-Ethoxycarbonylamino-6-(4-chlorophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-162 Ethoxycarbonylamino-6-(2-methylphenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-163 2-Ethoxycarbonylamino-6-(3-methylphenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-164 2-Ethoxycarbonylamino-6-(4-methylphenoxy)-3H-
imidazo[4,5-b]pyridine
A-165 2-Ethoxycarbonylamino-6-(2-fluorophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-166 Ethoxycarbonylamino-6-(3-fluorophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-167 2-Ethoxycarbonylamino-6-(4-fluorophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-168 Ethoxycarbonylamino-6-[2
(trifluoromethyl)phenylmercapto]-3H-imidazo[4,5-
b]pyridi ne
A-169 Ethoxycarbonylamino-6-[3
(trifluoromethyl)phenylmercapto]-3H-imidazo[4,5-
b]pyridi ne
A-170 Ethoxycarbonylamino-6-[4-
(trifluoromethyl)phenylmercapto]-3H-imidazo[4,5-
b]pyridine
A-171 Ethoxycarbonylamino-6-(2-
methoxyphenylmercapto)-3H-imidazo[4,5-b]pyridine
A-172 Ethoxycarbonylamino-6-(3-
methoxyphenylmercapto)-3H-imidazo[4,5-b]pyridine
A-173 2-Ethoxycarbonylamino-6-(4-
methoxyphenylmercapto)-3H-imidazo[4,5-b]pyridine
A-174 2-Ethoxycarbonylamino-6-(2-
carboxyphenylmercapto)-3H-imidazo[4,5-b]pyridine
A-175 2-Ethoxycarbonylamino-6-(3-
carboxyphenylmercapto)-3H-imidazo[4,5-b]pyridine
A-176 2-Ethoxycarbonylamino-6-(4-
carboxyphenylmercapto)-3H-imidazo[4,5-b]pyridine
______________________________________
Compounds A-177-A-198:
By substituting the appropriate aniline salt for sodium phenolate in
Example A-4, the identical process gives the following examples. For
examples A-196, A-197, and A-198, the carboxy groups are protected by a
methyl, ethyl, benzyl, t-butyl or other suitable ester and then
deprotected in the last step to give the compounds.
______________________________________
A-177 2-Ethoxycarbonylamino-6-phenylamino-3H-
imidazo[4,5-b]pyridine
A-178 2-Ethoxycarbonylamino-6-(2-nitrophenylamino)-3H-
lmidazo[4,5-b]pyridine
A-179 2-Ethoxycarbonylamino-6-(3-nitrophenylamino)-3H-
imidazo[4,5-b]pyridine
A-180 2-Ethoxycarbonylamino-6-(4-nitrophenylamino)-3H-
imidazo[4,5-b]pyridine
A-181 2-Ethoxycarbonylamino-6 (2-chlorophenylamino)-3H-
imidazo[4,5-b]pyridine
A-182 2-Ethoxycarbonylamino-6-(3-chlorophenylamino)-3H-
imidazo[4,5-b]pyridine
A-183 2-Ethoxycarbonylamino-6-(4-chlorophenylamino)-3H-
imidazo[4,5-b]pyridine
A-184 2-Ethoxycarbonylamino-6-(2-methylphenylamino)-3H-
imidazo[4,5-b]pyridine
A-185 2-Ethoxycarbonylamino-6-(3-methylphenylamino)-3H-
imidazo[4,5-b]pyridine
A-186 2-Ethoxycarbonylamino-6-(4-methylphenoxy)-3H-
imidazo[4,5-b]pyridine
A-187 2-Ethoxycarbonylamino-6-(2-fluorophenylamino)-3H-
imidazo[4,5-b]pyridine
A-188 2-Ethoxycarbonylamino-6-(3-fluorophenylamino)-3H-
imidazo[4,5-b]pyridine
A-189 2-Ethoxycarbonylamino-6-(4-fluorophenylamino)-3H-
imidazo[4,5-b]pyridine
A-190 2-Ethoxycarbonylamino-6-[2-
(trifluoromethyl)phenylamino]-3H-imidazo[4,5-
b]pyridine
A-191 2-Ethoxycarbonylamino-6-[3-
(trifluoromethyl)phenylamino-3H-imidazo[4,5-
b]pyridine
A-192 2-Ethoxycarbonylamino-6-[4-
(trifluoromethyl)phenylamino]-3H-imidazo[4,5-
b]pyridine
A-193 2-Ethoxycarbonylamino-6-(2-methoxyphenylamino)-
3H-imidazo[4,5-b]pyridine
A-194 2-Ethoxycarbonylamino-6-(3-methoxyphenylamino)-
3H-imidazo[4,5-b]pyridine
A-195 2-Ethoxycarbonylamino-6-(4-methoxyphenylamino)-
3H-imidazo[4,5-b]pyridine
A-196 2-Ethoxycarbonylamino-6-(2-carboxyphenylamino)-
3H-imidazo[4,5-b]pyridine
A-197 2-Ethoxycarbonylamino-6-(3-carboxyphenylamino)-
3H-imidazo[4,5-b]pyridine
A-198 2-Ethoxycarbonylamino-6-(4-carboxyphenylamino)-
3H-imidazo[4,5-b]pyridine
______________________________________
Example 2
Assay Measuring Phosphorylating Function of RAF
The following assay reports the amount of RAF-catalyzed phosphorylation of
its target protein MEK as well as MEK's target MAPK. The RAF gene sequence
is described in Bonner et al., 1985, Molec. Cell. Biol. 5: 1400-1407, and
is readily accessible in multiple gene sequence data banks. Construction
of the nucleic acid vector and cell lines utilized for this portion of the
invention are fully described in Morrison et al., 1988, Proc. Natl. Acad.
Sci. USA 85: 8855-8859.
Materials and Reagents
1. Sf9 (Spodoptera frugiperda) cells; GIBCO-BRL, Gaithersburg, Md.
2. RIPA buffer: 20 mM Tris/HCl pH 7.4, 137 mM NaCl, 10% glyercol, 1 mM
PMSF, 5 mg/L Aprotenin, 0.5% Triton X-100;
3. Thioredoxin-MEK fusion protein (T-MEK): T-MEK expression and
purification by affinity chromatography were performed according to the
manufacturer's procedures. Catalog# K 350-01 and R 350-40, Invitogren
Corp., San Diego, Calif.
4. His-MAPK (ERK 2); His-tagged MAPK was expressed in XL1 Blue cells
transformed with pUC18 vector encoding His-MAPK. His-MAPK was purified by
Ni-affinity chromatography. Cat# 27-4949-01, Pharmacia, Alameda, Calif. as
described herein.
5. Sheep anti mouse IgG: Jackson laboratories, West Grove, Pa. Catalog, #
515-006-008, Lot# 28563
6. RAF-1 protein kinase specflc antibody: URP2653 from UBI.
7. Coating buffer: PBS; phosphate buffered saline, GIBCO-BRL, Gaithersburg,
Md.
8. Wash buffer: TBST--50 mM Tris/HCL pH 7.2, 150 mM NaCl, 0.1% Triton X-100
9. Block buffer: TBST, 0.1% ethanolamine pH 7.4
10. DMSO, Sigma, St. Louis, Mo.
11. Kinase buffer (KB): 20 mM Tris/HCL pH 7.2, 150 mM NaCl, 0.1% Triton
X-100, 1 mM PMSF, 5 mg/L Aprotenin, 75 .mu.M sodium ortho vanadate, 0.5 MM
DTT and 10 mM MgCl.sub.2.
12. ATP mix: 100 mM MgCl.sub.2, 300 .mu.M ATP, 10 .mu.Ci .gamma.-.sup.33 P
ATP (Dupont-NEN)/mL.
13. Stop solution: 1% phosphoric acid; Fisher, Pittsburgh, Pa.
14. Wallac Cellulose Phosphate Filter mats; Wallac, Turku, Finnland.
15. Filter was solution: 1% phosphoric acid, Fisher, Pittsburgh, Pa.
16. Tomtec plate harvester, Wallac, Turku, Finnland.
17. Wallac beta plate reader # 1205, Wallac, Turku, Finnland.
18. NUNC 96-well V bottom polypropylene plates for compounds Applied
Scientific Catalog # AS-72092.
Procedure
All of the following steps were conducted at room temperature unless
specifically indicated.
1. ELISA plate coating: ELISA wells are coated with 100 .mu.L of Sheep anti
mouse affinity purified antiserum (1 .mu.g/100 .mu.L coating buffer) over
night at 4.degree. C. ELISA plates can be used for two weeks when stored
at 4.degree. C.
2. Invert the plate and remove liquid. Add 100 .mu.L of blocking solution
and incubate for 30 min.
3. Remove blocking solution and wash four times with wash buffer. Pat the
plate on a paper towel to remove excess liquid.
4. Add 1 .mu.g of antibody specific for RAF-1 to each well and incubate for
1 hour. Wash as described in step 3.
5. Thaw lysates from RAS/RAF infected Sf9 cells and dilute with TBST to 10
.mu.g/100 .mu.L. Add 10 .mu.g of diluted lysate to the wells and incubate
for 1 hour. Shake the plate during incubation. Negative controls receive
no lysate. Lysates from RAS/RAF infected Sf9 insect cells are prepared
after cells are infected with recombinant baculoviruses at a MOI of 5 for
each virus, and harvested 48 hours later. The cells are washed once with
PBS and lysed in RIPA buffer. Insoluble material is removed by
centrifugation (5 min at 10 000.times.g). Aliquots of lysates are frozen
in dry ice/ethanol and stored at -80.degree. C. until use.
6. Remove non-bound material and wash as outlined above (step 3).
7. Add 2 .mu.g of T-MEK and 2 .mu.g of His-MAEPK per well and adjust the
volume to 40 .mu.L with kinase buffer. Methods for purifying T-MEK and
MAPK from cell extracts are provided herein by example.
8. Predilute compounds (stock solution 10 mg/mL DMSO) or extracts 20 fold
in TBST plus 1% DMSO. Add 5 .mu.L of the prediluted compounds/extracts to
the wells described in step 6. Incubate for 20 min. Controls receive no
drug.
9. Start the kinase reaction by addition of 5 .mu.L ATP mix; Shake the
plates on an ELISA plate shaker during incubation.
10. Stop the kinase reaction after 60 min by addition of 30 .mu.L stop
solution to each well.
11. Place the phosphocellulose mat and the ELISA plate in the Tomtec plate
harvester. Harvest and wash the filter with the filter wash solution
according to the manufacturers recommendation. Dry the filter mats. Seal
the filter mats and place them in the holder. Insert the holder into
radioactive detection apparatus and quantify the radioactive phosphorous
on the filter mats.
Alternatively, 40 .mu.L aliquots from individual wells of the assay plate
can be transferred to the corresponding Positions on the phosphocellulose
filter mat. After air-drying the filters, put the filters in a tray.
Gently rock the tray, changing the wash solution at 15 min intervals for 1
hour. Air-dry the filter mats. Seal the filter mats and place them in a
holder suitable for measuring the radioactive phosphorous in the samples.
Insert the holder into a detection device and quantify the radioactive
phosphorous on the filter mats.
IC.sub.50 values were measured according to the protocol for the following
azabenzimidazole-based compounds in the RAF-1 ELISA assay:
##STR5##
An IC.sub.50 value is the concentration of the azabenzimidazole-based
inhibitor required to decrease the maximum amount of phosphorylated target
protein or cell growth by 50%. The IC.sub.50 values measured in the RAF-1
phosphorylation assay are depicted in Table 1:
TABLE 1
______________________________________
Compound
IC.sub.50 (.mu.M)
______________________________________
A-1 79
A-2 >100
A-3 6.1
A-4 6.7
A-90 0.95
______________________________________
Example 3
Purification of MAPK and MEK
The MAPK and MEK proteins are readily expressed in cells by subcloning a
gene encoding these proteins into a commercially available vector that
expresses the proteins with a poly-Histidine tag. Genes encoding these
proteins are readily available from laboratories that normally work with
these proteins or by cloning these genes from cells containing cDNA
libraries. The libraries are readily commercially available and a person
skilled in the art can readily design nucleic acid probes homologous to
cDNA molecules encoding MEK or MAPK from the nucleic acid sequences of MEK
and MAPK, available in multiple gene data bases such as Genbank. The
cloning of a gene can be accomplished in a short time period using
techniques currently assailable to persons skilled in the art.
Purification of the MEK and MAPK proteins from cell extracts can be
accomplished using the following protocol, which is adapted from Robbins
et al., 1993, J. Biol. Chem. 268: 5097-5106;
1. Lyse cells by sonication, osmotic stress, or French Press techniques
readily available to persons skilled in the art. An appropriate sonication
buffer is provided below.
2. Equilibrate a solid support which is conjugated with nickel or cobalt
with the equilibration buffer disclosed below. The poly-histidine tag
specifically binds to the nickel and cobalt atoms on the solid support.
Equilibration can be achieved by washing the resin three times with a
volume of the equilibration buffer equal to ten times the volume of the
solid support. The solid support is readily available to persons of
ordinary skill in the art.
3. Add the cell lysate to the solid support and equilibrate in a vessel for
a period of time. Alternatively, the solid support can be packed within a
protein chromatography column and the lysate may be flowed through the
solid support.
4. Wash the solid support with the wash buffer disclosed below.
5. Elute the MEK or MAPK protein from the solid support with an amount of
elution buffer (provided below) that removes a significant portion of the
protein from the solid support.
Sonication Buffer
50 mM sodium phosphate pH 8.0
0.3 M sodium chloride
10 mM .beta.-mercaptoethanol
1% NP40
10 mM NaF
0.5 mM Pefablock
Equilibration Buffer
50 mM sodium phosphate pH 8.0
0.3 M sodium chloride
10 mM .beta.-mercaptoethanol
1% NP40
10 mM NaF
1 mM Imidazol
Wash Buffer
50 mM sodium phosphate pH 8.0
0.3 M sodium chloride
10 mM .beta.-mercaptoethanol
1% NP40
10 mM NaF
10 mM Imidazol
Elution Buffer
50 mM sodium phosphate pH 8.0
0.3 M sodium chloride
10 mM .beta.-mercaptoethanol
1% NP40
10 mM NaF
10-500 mM Imidazol
Example 4
Assay Measuring Phosphorylating Function of EGF Receptor
EGF Receptor kinase activity (EGFR-NIH3T3 assay) in whole cells was
measured as described in detail in PCT Publication WO9640116, filed Jun.
5, 1996, by Tang et al., and entitled "Indolinone Compounds for the
Treatment of Disease," incorporated herein by reference in its entirety,
including any drawings.
The IC.sub.50 values measured in the EGF receptor phosphorylation assay are
depicted in Table 2:
TABLE 2
______________________________________
Compound
IC.sub.50 (.mu.M)
______________________________________
A-1 >100
A-2 >100
A-3 >100
A-4 >100
______________________________________
Example 5
Assay Measuring the Effect of Azabenzimidazole-Based Compounds on the
Growth of Cells Expressing RAS
The following assay measures growth rates for NIH-3T3 cells expressing RAS.
The purpose of the assay is to determine the effects of compounds on the
growth of NIH 3T3 cells over expressing H-Ras.
Materials
96-well flat bottom sterile plates
96-well round bottom sterile plates
sterile 25 mL or 100 mL reservoir
pipets, multi-channel pipetman
sterile pipet tips
sterile 15 mL and 50 mL tubes
Reagents
0.4% SRB in 1% acetic acid
10 mM Tris base
10% TCA
1% acetic acid
sterile DMSO (Sigma)
compound in DMSO (100 mM or less stock solution)
Trypsin-EDTA (GIBCO BRL)
Cell line:
3T3/H-Ras (NIH 3T3 clone 7 cells expressing genomic fragment of oncogenic
H-Ras).
The cells can be constructed using the following protocol:
1. Subclone a gene fragment encoding Ras into a commercially available
vector that will stably transfect NIH-3T3 cells. The fragment is from the
genomic transforming allele of cHa-ras.
2. Transfect NIH-3T3 cells with the subcloned vector by a calcium phosphate
method. Select cells expressing the Ras construct in 2% serum in DMEM.
Visible foci are observed after 2 weeks. Pool the transformed cells to
generate a stably transformed cell line.
Growth medium:
2% calf serum/DMEM+2 mM glutamine, Pen/Strep
Protocol:
Day 0: Cell Plating:
This part of assay is carried out in a laminar flow hood.
1. Trypsinize cells. Transfer 200 .mu.L of cell suspension to 10 mL of
isotone. Count cells with a Coulter Counter.
2. Dilute cells in growth medium to 60,000 cell/mL. Transfer 100 .mu.L of
cells to each well in a 96-well flat bottom plate to give 6000 cells/well.
3. Use half of plate (4 rows) for each comound and quadruplicate wells for
each comound concentration, and a set of 4 wells for medium control.
4. Gently shake plates to allow for uniform attachment of the cells.
5. Incubate the plates at 37.degree. C. in a 10% CO.sub.2 incubator.
Day 1: Addition of Compound:
This part of assay is carried out in a laminar flow hood.
1. In a 96-well round bottom plate, add 120 .mu.L of growth medium
containing 2.times. final % DMSO found in highest screening concentration
of compound to columns 1 to 11. For example, if the highest concentration
is 100 .mu.L, and this is made from a 100 mM stock, 1.times. DMSO is 0.1%
so 2.times. DMSO is 0.2%. This plate is used to titrate out the compound,
4 rows per compound.
2. In a sterile 15 mL tube, make a 2.times. solution of the highest
screening concentration of compound in growth medium plus 2.times. DMSO. 1
mL per cell line is needed. The starting concentration of the compound is
usually 100 .mu.M but this concentration may vary depending upon the
solubility of the compound.
3. Transfer 240 .mu.L of the 2.times. starting compound solution to
qudruplicate wells in column 12 of the 96-well round bottom plate. Do 1:2
serial dilutions across the plate from right to left by transferring 12
.mu.L from column 12 to column 11, column 11 to 10 and so on through
column 2. Transfer 100 .mu.L of compound dilutions, and 100 .mu.L of
medium in column 1, onto 100 .mu.L medium on cells in corresponding wells
of 96-well flat bottom plate. Total volume per well should be 200 .mu.L.
4. Return the plate to the incubattor and incubate for 3 days.
Day 4: Development of Assay
This party of assay is carried out on the bench.
1. Aspirate or pour off medium. Add 200 .mu.L cold 10% TCA to each well to
fix cells. Incubate plate for at least 60 min. at 4.degree. C.
2. Discard TCA and rinse wells 5 times with tap water. Dry plates upside
down on paper towels.
3. Stain cells with 100 .mu.L/well 0.4% SRB for 10 min.
4. Pour of SRB and rinse wells 5 times with 1% acetic acid. Dry plates
completely upside down on paper towels.
5. Solubilize dye with 100 .mu.L/well 10 mM Tris base for 5-10 min. on
shaker.
6. Read plates on Dynatech ELISA Plate REader at 570 nm with reference at
630 nm.
Select compounds inhibited the growth rate of cells over-expressing RAS as
illustrated in Table 3.
TABLE 3
______________________________________
IC.sub.50 (.mu.M)
Compound RAS/NIH3T3
______________________________________
A-3 40
A-4 20
______________________________________
Example 6
Assay Measuring Effect of Azabenzimidazole-Based Compounds on Growth of
A549 Cells
The following assay measures growth rates for A549 cells. The purpose of
the assay is to determine the effects of compounds on the growth of A549
human lung carcinoma cells. A549 cells are readily accessible from
commercial sources, such as ATCC (CCL185).
Materials:
96-well flat bottom sterile plates
96-well round bottom sterile plates
sterile 25 mL or 100 mL reservoir
pipets, multi-channel pipetman
sterile pipet tips
sterile 15 mL and 50 mL tubes
Reagents:
0.4% SRB in 1% acetic acid
10 mM Tris base
10% TCA
1% acetic acid
sterile DMSO (Sigma)
compound in DMSO (100 mM or less stock solution)
Trypsin-EDTA (GIBCO BRL)
Cell line and growth medium:
A549 human lung carcinoma cells (ATCC CCL185)
10% fetal calf serum in Ham's F12-K
Protocol:
Day 0: Cell Plating:
This part of assay is carried out in a laminar flow hood.
1. Trypsinize cells. Transfer 200 .mu.L of cell suspension to 10 mL of
isotone. Count cells with a Coulter Counter.
2. Dilute cells in growth medium to 20,000 cell/mL. Transfer 100 .mu.L of
cells to each well in a 96-well flat bottom plate to give 2000 cells/well.
3. Use half of plate (4 rows) for each comound and quadruplicate wells for
each comound concentration, and a set of 4 wells for medium control.
4. Gently shake plates to allow for uniform attachment of the cells.
5. Incubate the plates at 37.degree. C. in a 10% CO2 incubator.
Day 1: Addition of Compound:
This part of assay is carried out in a laminar flow hood.
1. In a 96-well round bottom plate, add 120 .mu.L of growth medium
containing 2.times. final % DMSO found in highest screening concentration
of compound to columns 1 to 11. For example, if the highest screening
concentration is 100 .mu.L, and this is made from a 100 mM stock, 1.times.
DMSO is 0.1% so 2.times. DMSO is 0.2%. This plate is used to titrate out
the compound, 4 rows per compound.
2. In a sterile 15 mL tube, make a 2.times. solution of the highest
screening concentration of compound in growth medium plus 2.times. DMSO. 1
mL per cell line is needed. The starting concentration of the compound is
usually 100 .mu.M but this concentration may vary depending upon the
solubility of the compound.
3. Transfer 240 .mu.L of the 2.times. starting compound solution to
qudruplicate wells in column 12 of the 96-well round bottom plate. Do 1:2
serial dilutions across the plate from right to left by transferring 120
.mu.L from column 12 to column 11, column 11 to 10 and so on through
column 2. Transfer 100 .mu.L of compound dilutions, and 100 .mu.L of
medium in column 1, onto 100 .mu.L medium on cells in corresponding wells
of 96-well flat bottom plate. Total volume per well should be 200 .mu.L.
4. Return the plate to the incubattor and incubate for 3 days.
Day 5: Development of Assay
This part of assay is carried out on the bench.
1. Aspirate or pour off medium. Add 200 .mu.L cold 10% TCA to each well to
fix cells. Incubate plate for at least 60 min. at 4.degree. C.
2. Discard TCA and rinse wells 5 times with tap water. Dry plates upside
down on paper towels.
3. Stain cells with 100 .mu.L/well 0.4% SRB for 10 min.
4. Pour off SRB and rinse wells 5 times with 1% acetic acid. Dry plates
completely upside down on paper towels.
5. Solubilize dye with 100 .mu.L/well 10 mM Tris base for 5-10 min. on
shaker.
6. Read plates on Dynatech ELISA Plate Reader at 570 nm with reference at
630 nm.
Select compounds inhibited the growth rate of A549 cells, as illustrated in
Table 4.
TABLE 4
______________________________________
IC.sub.50 (.mu.M)
Compound A549
______________________________________
A-3 22
65 (2% FBS)
A-4 6
5.7 (2% FBS)
______________________________________
Example 7
Method for Determining the Biological Activity of RAF Modulates In Vivo
Xenograft studies can be utilized to monitor the effect of compounds of the
invention upon the inhibition of ovarian, melanoma, prostate, lung and
mammary tumor cells. The protocol for the assay is described in detail in
PCT Publication WO9640116, filed Jun. 5, 1996, by Tang et al., and
entitled "Indolinone Compounds for the Treatment of Disease," incorporated
herein by reference in its entirety, including any drawings.
The invention illustratively described herein may be practiced in the
absence of any element or elements, limitation or limitations which is not
specifically disclosed herein. The terms and expressions which have been
employed are used as terms of description and not of limitation, and there
is no intention that in the use of such terms and expressions of excluding
any equivalents of the features shown and described or portions thereof,
but it is recognized that various modifications are possible within the
scope of the invention claimed. Thus, it should be understood that
although the present invention has been specifically disclosed by
preferred embodiments and optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled in the
art, and that such modifications and variations are considered to be
within the scope of this invention as defined by the appended claims.
One skilled in the art would readily appreciate that the present invention
is well adapted to carry out the objects and obtain the ends and
advantages mentioned, as well as those inherent therein. The molecular
complexes and the methods, procedures, treatments, molecules, specific
compounds described herein are presently representative of preferred
embodiments, are exemplary, and are not intended as limitations on the
scope of the invention. Changes therein and other uses will occur to those
skilled in the art which are encompassed within the spirit of the
invention are defined by the scope of the claims.
It will be readily apparent to one skilled in the art that varying
substitutions and modifications may be made to the invention disclosed
herein without departing from the scope and spirit of the invention.
All patents and publications mentioned in the specification are indicative
of the levels of those skilled in the art to which the invention pertains.
All patents and publications are herein incorporated by reference to the
same extent as if each individual publication was specifically and
individually indicated to be incorporated by reference.
The invention illustratively described herein suitably may be practiced in
the absence of any element or elements, limitation or limitations which is
not specifically disclosed herein. Thus, for example, in each instance
herein any of the terms "comprising", "consisting essentially of" and
"consisting of" may be replaced with either of the other two terms. The
terms and expressions which have been employed are used as terms of
description and not of limitation, and there is no intention that in the
use of such terms and expressions of excluding any equivalents of the
features shown and described or portions thereof, but it is recognized
that various modifications are possible within the scope of the invention
claimed. Thus, it should be understood that although the present invention
has been specifically disclosed by preferred embodiments and optional
features, modification and variation of the concepts herein disclosed may
be resorted to by those skilled in the art, and that such modifications
and variations are considered to be within the scope of this invention as
defined by the appended claims.
In addition, where features or aspects of the invention are described in
terms of Markush groups, those skilled in the art will recognize that the
invention is also thereby described in terms of any individual member or
subgroup or members of the Markush group. For example, if X is described
as selected from the group consisting of bromine, chlorine, and iodine,
claims for X being bromine and claims for X being bromine and chlorine are
fully described.
Those references not previously incorporated herein by reference, including
both patent and non-patent references, are expressly incorporated herein
by reference for all purposes. Other embodiments are within the following
claims.
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